This is based on opinion. Please don't list it on a work's trope example list.
Tear Jerker / Spider-Man: The Animated Series
After finally being freed from his imprisonment outside of the normal spacetime, Captain America willingly traps himself and the Red Skull again. And this time, there may be no way to free him again...
Eddie Brock getting sucked into a black hole like vortex, just after saving the only person he's ever cared about more than himself, ouch...
The death of Mary Jane's clone is pretty heart-wrenching, as is Spidey's Big "NO!" that follows it. Christopher Daniel Barnes gave his all in that scene.
This also makes it the SECOND time that Spider-Man watches her die. The first one's heartbreak doesn't have time to set in before the Nightmare Fuel of Spider-Man legitimately ready to kill the Green Goblin.
Spider-Carnage's talk with the still-alive Uncle Ben from another reality to talk Spider-Carnage out of destroying the multiverse. The speech moves him so much he actually starts crying, and subsequently performs a Heroic Sacrifice after realizing the Carnage symbiote still won't let him go and it hurts so much to try.
Spider-Man takes it pretty hard in "The Mutant Agenda" when Professor Xavier informs him that he can't help with his Neogenic disease. After Spidey swings off to angst, Beast tracks him down to suggest a place he can go for help.
Spider-Man: Why are you going out of your way for me? Beast: No man can turn his back on another's pain. I may be a mutant, but I'm still human. Spider-Man: Well, that's where we're different. I'm not sure I still am. [swings off] Beast: How sad. We are all alone until we accept our need for others.