Gosalyn singing "Little Girl Blue", the song her late Grandfather used to sing to her which is also the code to activate a powerful machine and Darkwing's verse afterward.
The scene where Gosalyn is being taken by Bulba's men and D.W. is desperately trying to save her while shouting her name.
Darkwing's Disney Death at the end of the episode. Sure, seeing as how there's nearly 90 more episodes after this you know that everything's going to be okay, but that scene where Gosalyn's forlornly staring at the spot where the Ramrod blew up with him on it from her room at the orphanage is heartbreaking.
Consider the fact that Gos' VA, the now late Christine Cavanaugh, gave up her career at the dawn of the 2000's to focus on her family. Now that she's gone, try listening to the lullaby without crying.
Up, Up, And Awry
When Gizmoduck and DW are stuck on a giant magnet and the former tells the latter that without his Powered Armor, he's useless. Yes, it does lead to a Moment of Awesome for DW followed by a happy ending, but it's still quite heartbreaking that even after all this time, Fenton STILL doesn't think he's worth anything without the Gizmosuit.
Gos and Launchpad returning from Darkwing's funeral.
Darkwing, who had died and is currently in a ghost form, saying goodbye to Morgana, Launchpad, and Gosalyn. Just the sight of not just Launchpad and Morgana crying, but Gos crying breaks your heart. Of course, it turns out to be a dream.
Gosalyn tries to tag along on mission, and Darkwing tells her to stay with the Ratcatcher. When she protests, he replies "I'm serious Gos, if something were to happen to you, I... (voice catches) I don't know what I'd do."
To see how Dark Warrior Duck has become so corrupted by his own sense of justice because the one person he loved (more than himself) was gone, and to see her return after all these years has a flood of bottled up emotions just flowing out. It was only worse when he felt betrayed by her near the end as well.
The alternate Launchpad. He tried so hard to help Darkwing after Gosalyn's disappearance, and ultimately Darkwarrior wound up hating him enough to want to kill him. The fact that Launchpad doesn't hate Darkwarrior in return makes it even worse — as Darkwing's old friend, he still misses and loves him, but Darkwarrior is just too far gone.
When Darkwing and Gosalyn find Launchpad and find out that he’s going to ‘Oompa [marry]’ and leave Earth, Gosalyn cries about how she’s going to miss Launchpad and how no one could ever take his place. Even Darkwing looks sad at the thought of Launchpad leaving.
In issue #4, it looks like Gosalyn has bit the big one and DW cradles her body whispering "This can't happen. We can't let Bulba have this. It was you...you inspired me to come back. You've always been the inspiration. Your courage. Your honor. Having you by my side made me unstoppable. You were always my superhero." OK, we all knowshe wasn't going to die but still...
Darkwing trying to break the brainwashing on the catatonic Morgana in issue 5 of the comic. He succeeds, taking the entire issue to do so, also revealing that without Darkwing, Drake is a duck without a driving purpose. In the end, just about to give up, he tearfully says to her that if he never quit, she would never have suffered. The tear breaks her out of it.
The ending of the Quackerjack story in the Darkwing Duck 2011 Annual is on equal with an ending of Batman: The Animated Series. Quackerjack builds a ray to turn people into dolls. DW finds a Mr. Banana Brain doll online and someone snipes it off him from ebay. It turns out it was QJ's girlfriend in hopes of reforming QJ as he had a normal life before going totally crazy at the time DW got back in the game. Through a long manner, both the original Banana Brain and his new Banana Brain come alive and fight over him and DW breaks them up. QJ goes up to his girlfriend's house, turns himself into a doll and leaves a note saying "This is the best I'll ever be".
In the comics DW actually throws Launchpad out of his house after LP accidentally leads Nega-Duck to their home. This is sad in itself as this action destroys the little family they made. They got better, but during the time apart, LP thought he was tossed out because DW lost a contest. That means that when DW kicked him out, he didn't even tell him WHY his friend was throwing him out of his home. And while we knew LP could crash at Scrooge's place it's not clear that DW knew, so for all he knew DW was throwing his friend out onto the street, homeless AND jobless. Yet LP seems to hold no real grudge and is just happy to be back with his family when they reunite.
With the Dangerous Currency arc made noncanon by the omnibus, until the new series, Morgana is still missing. Quackerjack is also likely still a doll, though it's almost a given that the new series will devote some issues to bringing both of them back.
In issue #8 of the Joe Books run, Bushroot is forced to kill Posey with his own powers after she's turned practically everyone in St. Canard into potato zombies yet again, and despite his best efforts to talk her down peacefully.