* [[spoiler:Hex]]'s HeroicSacrifice in Season 4.
** [[spoiler: "Take care of him for me..."]] *sniffle*
* The ending of "Web World Wars", after [[spoiler:Bob is shot into the web.]]
* Two moments from "Game Over":
** Megabyte orders his fleet to attack the principal office. The fleet is cut off and destroyed, yet the viral binomes go down fighting, the commander shouting his loyalty to Megabyte. Megabyte, huge JerkAss that he is, is unaffected, but [[WhatASenselessWasteOfHumanLife Dot weeps over the slaughter]].
** The ending. Made even worse by everybody's shocked silence (19 seconds, of everyone in the War Room just shocked and staring at Dot in disbelief and even mild incomprehension) after they hear the words [[spoiler:"GAME OVER. USER WINS."]], only to have it broken by Dot's [[BigNo grief-stricken scream]].
** Surprisingly, Slash gets a minor moment when he laments the way things have been going in Mainframe lately.
--->"Bob always stopped us before we could do anything ''really'' bad. Now, nobody does."
* The moment where Enzo, [[spoiler: now Matrix, is reunited with Dot]] is a tear-jerker of a [[CrowningMomentOfHeartwarming heartwarming sort.]]
-->''"Uh, hi..... sis."''
* One thing about Matrix and Dot's reunion that always gets this troper is the short look of confusion on Dot's face. Either she doesn't recognize her brother at first or she's in denial that it's really him and it HURTS to see.
** It doesn't help that Matrix grew up at an accelerated speed so he's not supposed to be anywhere near as old - something that almost certainly hit Dot.
* Hex curled up in the Fetal position and shrinking away from Scuzzy at the end of "Painted Windows".
* Hex's reaction to her masks being fused together, effectively bringing her full sanity for the first time in her life.
-->'''Bob:''' How do you feel?
-->'''Hex:''' For the first time... WHOLE.
* Though it leads to a HappyEnding, in ''End Prog'' the forced system crash Bob implements by allowing the User to win a Game by keeping everyone out of it. The reactions from the Sprites and Biomes as their world is ending all around is devastating, especially an image of a Little Girl Biome, all alone without her parents or anyone, cradling her doll as the System Crash is announced.