This is based on opinion. Please don't list it on a work's trope example list.
Tear Jerker / Who Framed Roger Rabbit
Marvin Acme's death. That's all you need to know.
Basically, anything having to do with the murder of Teddy Valiant and Eddie's memory of him. It's the heart of the film, after all, and is what transformed Eddie into a grumpy, alcoholic, washed-up has-been in the eyes of others. The only reason Roger comes to his aid at all is because he seemingly never realizes Eddie's changed, and is under the impression he still helps Toons.
The scene in the movie theater signals the start of the bond between Eddie and Roger. It's the first time we hear of how Teddy died... and foreshadows Judge Doom's real identity. Roger, heart-broken, and convinced that Eddie hates him and wants to kill him, may be hammy, but still tugs at the heart strings.
Roger crying, period. He's such a sweetheart. When he cries, our hearts ache.
Hell, even the scene of Eddie alone in his office, when the camera pans across the memorabilia of the brothers' successful cases.
Especially because the shoe was so innocent and acted like a little defenseless puppy... and, good God, the sounds it made when Judge Doom dipped it!
It was snuggling up next to his shoe! The poor little thing just wanted to be friends!
You know, that there is another shoe out there that will always be alone now...
Seeing Roger sobbing desperately after he believes Jessica has cheated on him with Marvin Acme, especially when he pulls out pictures of their wedding and honeymoon. To see Roger, normally so cheerfully wacky, obviously completely heartbroken really hits you...
In a meta way, Eddie's line, "Who needs a car in LA? We've got the best public transport system in the world," when you compare it to the gridlock of today and understand that the auto industry deliberately dismantled LA's streetcar system to force people to buy cars.
Jessica's line about how difficult it is to go around looking the way she does. If the hooting in the nightclub is any evidence, she doesn't get much respect.
Alternately, other toons don't take her seriously because she's sexy- and sexy isn't funny. There's a reason she's considered the lucky one for snagging Roger as a husband by other toons such as Betty Boop.
Or, perhaps, all of the aboveat once. Poor Jessica's got it pretty bad regardless of how you interpret her statement, really.
Toontown is home to all toons, no matter the animation studio. If Judge Doom had succeeded, Snow White, Mickey, Donald Duck, and every other Disney character kids love—-along with the likes of characters such as Bugs Bunny and Daffy Duck—-would be dead forever.
Even though he was a greedy, manipulative Jerkass, R.K. Maroon did care for Toons to some degree. The moment he passionately declared "I've been around Toons all my life, I didn't wanna see 'em destroyed", explaining why he wanted Acme's Will could make him a Jerk with a Heart of Gold.
The way he cries out when he's shot in the back, he'd thought Eddie shot him.
If you know Betty Boop's history in regards to her anthropomorphic dog boyfriend, (short explanation, her boyfriend Bimbo was changed to being her pet when the Hays Code deemed their relationship to be a promotion of bestiality) then the part where she is staring wistfully at Jessica as she comments on how lucky Jessica is might tug at your heart strings a little.
Likewise, crossing over into a meta-tearjerker, we get the fact that Boop is also working a cigarette girl in a nightclub, as she had been put out of business by cartoons going to color.note She has one cartoon in color, Poor Cinderella (1934), where she plays a Significant Greeneyed Redhead in it.. Granted, while she isn't too upset about it, although she does assure Eddie that she still has it, to which he replies "Sure, Betty." One could think this movie for reviving her career, albeit as a merchandising icon.