A subtle one for me, but when Agatha stumbled onto Dr. Dimitri's teddy bear stash, and he's begging her not to tell..."He doesn't know! It's my last secret! He's taken all the others, but not them! I've kept them safe!" Seeing the scars on his head, and then finding out that Baron Wulfenbach has been lobotomizing Sparks to see how to deactivate them makes it particularly sad to this troper...
What makes it even worse is imagining what would happen if Wulfenbach found out about the stuffed bears. No matter what Klaus' reaction would be (either to let him keep them or take them away) it would be heartrending.
Gets worse in the novelization - when they're escaping Castle Wulfenbach, Krosp drags Agatha and Theo off to find him. When they do, Vapnoople reveals that the bears are supposed to be his new army, but though he still acts like a Spark, he isn't one anymore, and they're forced to leave without him.
Krosp calls Vapnoople Papa.
How about in "Agatha vs the Jägers"? Seeing a superhuman immortal hunter monster collapse into tears because he found the Mistress he loves and swore to obey, plus gets to go home? Their cause was literally hopeless...
Ognian: Ve haff missed you! Please, please be real! Agatha: Shh. I... I am.
Othar's Twitter, of all places, has one of the sweetest and saddest relationships ever.
Thesetwo pages, as Barry gives Agatha her locket. Made even worse by the fact that Barry Heterodyne, who freaks out even Lucrezia Mongfish, the OTHER, is crying. And that young Agatha seems to be just as intelligent as she should be, rationalizing Barry's statement of "it's science" to "Ah. You mean you'll explain when I have a sufficiently advanced background education." Necessary or not, what Barry did to Agatha was horrible.