- Pretty much the entirety of Deadpool's level, especially when he's talking about Pete's love life.
- Ow, my uterus!
- "Listen, Webby, I can't have you swinging around in your footy pajamas, destroying my cameras. You're going to have to tangle with my army! And by army, I mean production assistants, and by production assistants, I mean unpaid interns, and by unpaid interns, I mean fans. You're going to have to tangle with my fans!"
- 2099 Spidey goading on Doctor Octopus by responding to her scientific marvels with a simple "Meh" to the point where she breaks down into a furious rage is hysterical.
- This from Vulture's stage:
Thug #1: If Spider-Man shows up here, I'll fill 'im fulla daylight.
Thug #2: Pfft. It's nighttime.
Thug #1: The same principle applies!
Thug #3: But it don't sound as good. "I'm gonna fill you full of nightlight?"
Thug #2: See, this is why we're just goons. We stand around havin' stupid discussions like this.
- During Electro's stage:
Spider-Man: (after hearing Electro make suggestive comments as he absorbs energy) Uh, eww! Do you and your generator, like, need some time alone?
- This gem from 2099's third stage.
Doc Ock: Computer, this lab has been compromised. Activate self-destruct sequence... ten seconds.
Spider-Man 2099: Ten seconds? That's barely enough time to come up with a clever explosion related quip.
Doc Ock: Ooh, that must really burn you up. So long, Spider-Man. It's been a blast.
Spider-Man 2099: Oh, she's good.
- Any interaction between 2099 and Doc Ock qualifies.
Spider-Man 2099: Ooh, I'm really getting under your skin! If I keep this up, you'll have a meltdown.
Doc Ock: I won't.
Spider-Man 2099: You will!
Doc Ock: Silence!
Doc Ock: "Behold my great invention!"
Spider-Man 2099: "…Meh."
'Doc Ock: "'Meh?' What do you mean, 'meh?'"
Doc Ock: "Whuh-no you haven't!"
- Within the first five minutes:
Madame Web: And now, scale the wall with your unique adhesive ability.
Amazing Spider-Man: I believe the technical term is "stick 'em powers".
(A couple minutes later in the Ultimate universe)
Madame Web: And you can run across narrow walkways such as this one without fear of falling, due to your natural agility and powers of adhesion.
Ultimate Spider-Man: They're called "stick 'em powers"... did one of the other me's make that joke already?
Madame Web: Yes, and it was no funnier then.
- "This quest involves jumping?!"
- One of Amazing's first observations is that he better get going, since this neighborhood isn't going to "Friendly" itself.
- Amazing Spider-Man's reaction to seeing the many different Spider-Men scattered across reality:
Amazing Spider-Man: Is that... a cartoon pig?
- Amazing Spider-Man in the DS version: Conveyor belt? Am I in a video game now?
- Amazing in the DS version before entering the final portal.
- The ending after Stan Lee's narration. What'd I miss?
- The end-credit gags involving the various villains, including Kraven trying and failing to catch a bunny, Goblin working as a carnival barker, and Scorpion's inability to use scissors.
- Doc Ock 2099 is shown as an Octomom to child versions of Hobgoblin and Scorpion.
- Ultimate, after Electro flings his fifth and sixth trucks at him:
Ultimate: You really have a thing for trucks, huh? Are you sure you want to be called Electro? How about Elec-truck? Truckodon? Truck Master! Truckleberry Finn! Trucktor Doom! Oh, I know! Professor Pickup! That is something else."
Electro: Be quiet!
Ultimate: I really drove that one into the ground!
Electro: Maybe these will shut you up!!
Ultimate: Or maybe it'll make me talk more! You never know...
- Ultimate, fighting Carnage
"I am not losing to a monster made of - of malevolent spaghetti! ...Did I just say that?"
- In the opening of the Juggernaut's level.
- His luck doesn't improve in the fight.
Okay, laughing boy, now hold still while I deliver the Coup de Grâce
Juggernaut: It means you're outta here! (chucks him through a building, causing him to land several blocks away)
Spider-Man: (pained) Well, I guess that's one interpretation.
- In the first Noir episode, some of the mook dialogue comes off as Seussian.
Mook 1: Did you hear about Fancy Dan?
Mook 2: Fancy Dan, the Ladies Man?
Mook 1: They found him in a garbage can.
Mook 2: Oh, man, Fancy Dan...
- When Spider-Man asks Kraven how he got the tablet fragment.
Kraven: I am not completely ignorant in the ways of modern man, my friend. I secured it on... the Internet!
Spider-Man: You're joking.
Kraven: No. I had the winning bid.
- There's something strangely funny about Hobgoblin's gibbering as he bombs the place. Plus, when Spider-Man 2099 kicks them back.
Hobgoblin: Pumpkin bombs, pumpkin bombs, PUMPKIN BOMBS FOR EVERYONE!
Spidey: No, thanks, you can keep this.