It's a minor thing but, the squashing of the grasshopper. Poor Johnny didn't mean to do it. And the resulting talk about death with Stephanie is when it suddenly dawns on him that when NOVA show up, they're basically going to kill him.
Johnny Five: Squash... dead. Disassemble... dead. Disassemble,DEAD?![immediately flees in terror]NO DISASSEMBLE!!
Johnny Five's apparent demise. Yes, it's a Disney Death, but still... Even Howard seemed pretty despondent, (and not just for losing tech worth millions of taxpayer dollars) just before firing Skroeder.
Stephanie begging and screaming for the armed forces not to shoot beforehand, insisting Johnny is alive. When all that's left is Johnny's supposed remains being picked off the ground, her mortified reaction sounds genuinely equivalent to seeing someone's disembowelled corpse scattered in several pieces.
Short Circuit 2:
During another reading binge at a bookstore, Johnny finds two books that he says he will read "very carefully". After he is arrested by the police, and treated as property rather than a person, he's shown reading one of the books slowly and carefully, rather than flipping through in seconds like he normally does. The two books? Frankenstein and Pinocchio, both about man-made creations who were brought to life, just like Johnny himself.
Johnny's understandably hurt reaction to learning Fred was going to sell him out (literally).
Johnny: Fred wants to sell ME?! Fred: (laughs sheepishly) Oh, J-5, c'mon, it's just a figure of speech. Johnny: Am not property, Frederick! One whose person is under control of another as master, is a SLAVE!
"DYING" It's not a spoken line; Johnny is trashed so badly that he has to scrape the word into a brick wall with a rock.
The look on Fred's face when he finally finds Johnny... and sees the condition he's in. His expression goes from joy to horror in seconds.
Johnny getting Fred's name wrong may seem funny, until you remember some of his brain is still damaged.
Fred: Are you okay? Johnny Five: Functioning 100%. Perfectly ko Derf. Fred: It's Fred. Johnny Five: That's what I said, Derf.
Near the end, when Benjamin tries to revive Johnny Five's battery power with a defibrillator, just after catching Oscar.
At one point, Ben ends up screaming at the heavens, "PLEEEESE! COME OOOOONNNN!!!"
Crosses over into Funny Moments, but after Ben revives Johnny, he threatens the paramedic who loaned him the defibrillator:
Ben: Keep that power on or I'll beat the living headlights out of you!
It is equal parts this and Nightmare Fuel, but during the beatdown he gets from the bank robbers, Johnny is screaming in pain and pleading for his life while Oscar continuously yells to the robbers to smash Johnny. One of the robbers (who was shown earlier to be a tech genius and borderline Minion with an F in Evil) hesitates for a second after getting sprayed with a spurt of battery fluid (that looks eerily blood-like), before committing himself to help in the smashing.
"No! Please, stop! I'm alive! Do not kill m" *smash!*
While it's also a Moment of Awesome, throughout Johnny's Roaring Rampage of Revenge, there's a constant cut to his ticking fuel timer, while his parts won't stop sparking from damage, you can even hear him yelp in agony. As cathartic as it is to see him give payback to his muggers, the whole thing still manages to be thoroughly heart wrenching.