* [[https://www.youtube.com/watch?v=nROpAhQ8pGo This]] DownerEnding from ''The Invisible Hand of Fate'': Sgt Hatred [[spoiler: is revealed as a high-ranking Guild mole in the OSI; Hunter is reassigned to Guam, Brock is reassigned to the Venture Compound as punishment for the failure of the BIlly-Phantom Limb-infiltration and explosion; Phantom Limb wakes up his now-superpowered self destined now for a long , though eventually ignominious career with the Guild; and Billy]] is delivered to a devastated Pete White. No other imagery in the show so far has so perfectly captured the theme of failure.
* Powerless In The Face of Death's ColdOpen show Rusty running away from home [[spoiler: after the boy's deaths]] in order to escape the pain of losing them. [[spoiler: More or less becomes a MoodWhiplash when it's revealed they've died many times before and Rusty keeps constant back up clones, but since he's run out of clones, who knows what will happen if Hank and Dean do die again?]]
* The scene in ''Everybody Comes To Hank's'' where [[spoiler: the identity of Dermott's actual father (and ''mother'', for that matter)]] is revealed. The [[TeenPregnancy situation]] is played heart-breakingly realistic and is only exacerbated by the music that accompanies it.
* 21 breaking down into sobs when he realizes [[spoiler: 24's ghost/figment of 21's own imagination]] is finally, truly gone.
* When Rusty sees an alien save him in the form of his dad, he believes his dad is truly alive. His face and tone display are truly heart-breaking.
* The ending of "Any Which Way But Zeus". Generally seen as [[HeartwarmingMoments a happy moment of friends reuniting]], but people often miss seeing the tall bearded man looking around desperately, and we see his chin start to shake from suppressed sobs right before the camera cuts away. Many people don't know what to make of it, [[FridgeHorror then you notice his costume and you realize who he is, Iron Patriot, and you remember the first gladiator fight seen in episode. 21 fighting Ghost Robot who is "working under the hero, Iron Patriot",]] and you remember 21 killed him!
** Although ''Bot Seeks Bot'' shows that [[NotQuiteDead he's alive. Somehow]].
* Ben's speech about why Doctor Venture chose to clone his sons is truly heartbreaking, especially for parents who lost a child.
-->'''Dean''': It's just...it's just so wrong!
-->'''Ben''': Have a kid one day, Dean. Hold its lifeless body in your arms, and then tell me how wrong it is. Your grandpap and I perfected genetic engineering because God or whatever gave us a perfect map to do so. Jonas, me, and, yes, your dad saw it as nothing more than a fucking Band-Aid for a really big boo-boo. [[MoodWhiplash Hey, you...you want a beer?]]
* Brock saying what ever he has to tell Hank and Dean to get them away from danger "The Family That Stays Together, Slays Together Part 1"
-->'''Dean''': But we're a family! We stick together through thick and thin!\\
'''Brock''': No. You guys are the family. I'm just a guy who was hired to protect you.\\
'''Hank''': But we... love each other...\\
'''Brock''': I ''don't'' love you boys! It was a job! Nothing more! Now that it's over, you'll only get in my way.
** Takes a turn for the [[CrowningMomentOfHeartwarming heartwarming]] and [[CrowningMomentOfFunny funny]] moments later [[spoiler:when it turns out Hank didn't buy it for a second, correctly dismissing it as the "Lassie trick".]]
* Those poor baby gorillas.
** Especially so when you see Hatred's face when he's forced to shoot the baby gorillas (after he had killed their parent gorilla). Immediately after that, as the team is setting camp in the bio dome, Hatred is briefly shown to still be affected by this.
* The Monarch's complete & utter denial that Gary quit. He's absolutely convinced that Gary is still his top Lieutenant, on a deep cover infiltration of the Venture Compound, even months later.
** Meanwhile, poor Gary has been pushed to the side when SPHINX joins up with OSI. He's not allowed to join OSI due to his criminal past, so he spends his days as the only inhabitant of SPHINX headquarters. And it's still better than working for the Monarch in his mind.
* [[spoiler:Dean burning his learning bed and his old things. The music was sad enough, but when he reaches for his stuffed Giraffe, you can tell he's not enjoying it.]] This line sells it.
-->'''Dean:''' [[spoiler: I'm sick of living my life in a box! I don't wanna be a scientist, or a boy adventurer, or even a Venture Brother anymore.]]
* The Monarch's freakout when he learns about a certain tidbit about his childhood.
** To elaborate, [[spoiler:The Monarch finds an old picture of himself as a child playing at the Venture Compound with Rusty with his parents and Jonas Venture, and he has absolutely no memory of this]]. Also from the same ep, the old SPHINX crew hijacks Gary's operation to get revenge on the OSI in a blaze of glory as their cyanide capsules will activate in a year. Especially with the old SPHINX commander losing his girlfriend and never fully moving on and another member having a wife and baby that he'll never see again.
* The whole reason behind why Spanakopita is both sad and pretty messed up. The whole event is [[spoiler:basically a StockholmSyndrome holiday]] since it was [[spoiler:only created to try and cheer up Rusty when he was a kid after they kidnapped him.]] [[spoiler:His kidnappers]] had a change of heart when they were utterly heartbroken at [[spoiler:Rusty's reaction]] after he discovered that [[spoiler:his Dad had never responded to their ransom.]] The kid was utterly devastated as he felt so alone and scared, [[spoiler:Rusty cried long and hard as his captors just couldn't stand to see a boy so sad.]] The saddest part of the thing is that Spanakopita is the only time that Doc ever acts or feels so truly, genuinely happy. Not even White has ever seen Doc so remarkably happy before in all of his life.
** Man, Rusty's dad's treatment to him can be a general downer. Not only does his Dad ditch him on a boat all by himself for some fancy wedding, but instead of coming back later that day the guy was gone for several days. I know he was off fighting bad guys and saving one of the Apollo's, but he never called his Son or anything like that. His dad just went off and just wanted to do his own thing like usual while he was utterly clueless at how much [[spoiler:his son was heartbroken as he was kidnapped with him not knowing tell much later on.]]
** Jonas wasn't just off doing his own thing. A friend of his was mentioned to have been on the Apollo's and the attempts weren't possible because the action lasted over several days as the kidnappers attempts at reaching him show. This arguably makes things worse as for once Jonas isn't being the willfully negligent jackass he usually was. Just further proof of how the boy adventurer life could be traumatic, even the positive parts of it could have long term circumstances.
*** Though you have to admit, he does get some points at the end when he [[spoiler: lets Rusty leave the room with his happy memories in tact before going PapaBear on his kidnappers]].
*** [[spoiler: Was that really for Rusty's benefit, or because they had the nerve to try and kidnap the son of the great Jonas Venture?]]
*** No, you really do have to give some points for waiting until the door closed [[spoiler: BEFORE going apeshit]].
* The ending of O.S.I Love You. Brock [[spoiler: attempts to kill off Molotov once and for all, ending his constant chase against a woman who will probably never love him for real and would prefer to just play mind game after mind game.]] Now, a lot of previous watchers of the Venture Bros knew [[spoiler: he wouldn't do it]], but what they didn't expect was [[spoiler: being ordered by his own comrades to let her go, revealing that she now works for the O.S.I because they're paying her more; after all, she's a mercenary.]] The sad thing is, it looked like Brock [[spoiler: was really going to do it]], and [[spoiler: Molotov offering him a handshake after the announcement]] feels like a punch in the gut for people who were really hoping Brock [[spoiler: could leave her behind]].
* In ''Bot Seeks Bot,'' Councilman #1's [[spoiler: last words are repeating "Where is my wife? I think the plane is going down." this is sad enough on its own, but it's also revealed that he was a cyborg built by Jonas Venture Sr. out of a friend of his who died in a plane crash. And The Monarch's parents ALSO (allegedly) died in a plane crash. And that picture of The Monarch with Rusty as a kid has his parents, Dr. Venture, and a plane. And yet The Monarch doesn't seem to recognize Vendata either...]]
* From ''The Devil's Grip'' (Season 5 Finale), there's [[spoiler: The Monarch's soul-crushing heart-to-heart with Rusty after [[TooKinkyToTorture giving up on trying to torture him]]. The two of them really do have a lot in common, and are both really put down, with Doc admitting that he'd rather have The Monarch keep torturing him than realize how similarly depressing they were. And even though The Monarch tells Dr. Mrs. The Monarch that it was all a ruse to crush Rusty's spirit, before [[LargeHam hamming it up about how he's "CRUSHED DR. VENTURE,]], his tone and returning to looking at the aforementioned picture make it clear it affected him as much as Rusty. Who, despite being a child star, is now hitchhiking home, with even his own bodyguard flying his Hover Tank above him in a RoaringRampageOfRevenge.]]
** And from the same episode, [[spoiler:Action Man bids farewell to Ant Men, who he accidentally killed with his rocking chair. He also admits to Hank that he's been trying to hook up with a woman named Rose, who turns out is Billy's mother. Meanwhile, Colonel Gentleman is saddened by all the friends he's lost over the years and how empty his home has become. All the adventures they've had, only to be so lonely.]]
** Also the boys' reaction to reading [[spoiler: Sgt. Hatred's note about Doc having most likely died, and that he's probably committing suicide and burning their house down, leaving the boys with their godfathers to learn everything all at once]].
* When the boys discover [[spoiler: their clones]], they go beyond a HeroicBSOD and collapse on the floor in a fetal position [[spoiler: probably realizing they are one of many sets of clones.]] Fortunately, Rusty comes up with a good cock-and-bull story explaining them as a "Christmas gift" that they spoiled, so he'll have to give him something better when Christmas rolls around, but there's genuine pathos with Rusty, Brock and the boys before he's able to lie to them.
* [[spoiler: Speedy's death in the first episode. Even though he brought it on himself by trying to take on Brock Samson, it's still really sad.And he was so close to getting his wings.]]
* [[spoiler: JJ's HeroicSacrifice in ''All This And Gargantua 2.'' Rusty looked ''genuinely'' heartbroken as he helplessly watched his brother float away to his death.]]
* Dr Orpheus losing his daughter to his ex and her new husband. Its pretty clear he misses her and can't get around to see her often.
* Gary's character arc in season 5. The very morning after he decides to quit working for the Monarch and work for SPHINX because GoodFeelsGood, he gets kicked off of SPHINX because everyone else went back to work for the OSI. He can't join them due to his past as a henchman so he spends his time patrolling the compound by himself in an empty warehouse. He screws up fighting bad guys and when tries to get recruits, he ends up recruiting the original, villainous SPHINX members and they perform a mutiny on him. At the end of that, he loses the SPHINX airship and, by accident, his warehouse base gets blown up. By the end of the season, he finally gets some long due respect from Sgt. Hatred, only to lose his friendship when they both get manipulated by the Moppets into becoming enemies again. This all ends in a ShaggyDogStory when he then goes back to being a Henchman again as he started missing his old life. The only positive thing about it is that [[spoiler:he finally avenged 24's death by rallying the other henchmen to presumably kill the Moppets, revealed to be the murderers]].