Gru, trying to flatter Ms. Hattie by speaking Spanish. She has no idea what he's saying, but she thinks it's a compliment. However...
"You have a face... como un burro."*
"You have a face that looks like a donkey."
And when Ms. Hattie buys a Spanish dictionary to notice what it means.
Margo's sarcasm when Vector orders cookies, Edith praying in the orphanage just to scare Agnes and asking Gru if the girls could touch the floor & the air.
Margo: "Four boxes of mini-mints, two toffee totes, uh, two caramel clumpies, aaaaand fifteen boxes of coconutties."
Vector: "Exactly. I'd like to see somebody else order that many cookies. Not likely. Name one person who ordered more cookies than me."
Edith: And bless that while we're sleeping, no bugs will crawl in our ears and lay eggs in our brains.
Margo: Great... Thanks for that image, Edith.
Gru: As you can see, I've provided everything a child might need. [puts down two dog bowls and a newspaper] Alright. Um, okay, now as I was saying... Hey! Oh! GGRRRR!!
Edith: Somebody broke that.
Gru: Okay, okay, clearly, we need to set some rules. Rule number one: you will not touch anything.
Margo: Uh-huh. What about the floor?
Gru: Yes, you may touch the floor.
Margo: What about the air?
Gru: Yes, you may touch the air.
The glowstick.
There's just something about Vector screwing around with the shrink ray, and the idiotic one-liners he shouts while shrinking his BATHROOM.
Vector: Booyah! You got shrunk, tiny mouth wash!
CURSE YOU TINY TOILET!
Also, what happens when Dr. Nefario mishears "cookie robots." Gru's expression just sells it.
Made even funnier by the end of the movie.
The girls messing with Gru's presentation. "I grab the moon... I sit on the toil...et- what?"
Gru:(to Dr. Nefario at the Boogie Robots presentation) 'Cookie robots.' I said 'cookie robots.' W...Why are you so...so old? Nefario: (beat) Ok. I'm on it.
Gru's facial expression and pointing over his back at hearing Vector was the one who stole the pyramids gets a giggle.
Early on in the movie, there's a game at an amusement park where you have to knock over a tiny spaceship prop. The reward is a stuffed unicorn (Agnes' was destroyed earlier). After around 1 or 2 tries, Agnes hits the spaceship... but the vendor decides to be a dick and invoke Exact Words, saying that she didn't "knock over" the spaceship. Gru proceeds to bring out his More Dakka gun, blast the stand, and yes, knock over the spaceship.
Even better: the entire stand is obliterated... except the planet and spaceship. The vendor stares at the wreckage, and the ship disintegrates.
"...who's Debbie?"
Agnes holding her breath.
Gru: ...that's freaking me out, what is she doing?
The little shadow puppets during the end credits can make one giggle.
Minion (talking through shadow puppet of Gru): "Hello! I am Gru. Back to work. Back to work!"
Agnes singing at the orphange while they were about to sleep.
Two of the Minions altering Gru's credentials while Ms. Hattie is reading them. Then getting into a slap fight over what to add, and then one Minion starts forcing the other to slam his head against the keyboard repeatedly, causing the credentials to turn to gibberish and confusing the hell out of Ms. Hattie.
"Yes, mine's shaped like a dead guy!"
One of the side effects of stealing the moon... a werewolf turns back to normal.
"Rule number three: You will not cry, or whine, or laugh, or giggle, or sneeze or burp or fart."
Agnes: "Does this count as annoying?" (puckers cheeks)
Gru: "VERY."
The minions playing with the copier.
"Butt."
The girls and the minions throwing TP around the house.
Gru's odd "good night" to the girls during their first night there.
"All right, good night, sleep tight, and don't let the bed-bugs bite. Because there are literally thousands of them." *turns off lights, starts to leave, but pauses* "Oh, and there's probably something in your closet."
That part with the balloon.
The minions go shopping for a new unicorn toy for Agnes.
"When we got adopted by a bald guy...I thought this would be more like Annie."
The Brick Joke in which Ms. Hattie mentions the offscreen box of shame, then when the three girls leave the room, they say hi to the girl imprisoned in the now-visible box.
Edith's face when she sees Gru for the first time.
Agnes eating candy from a dog bowl.
The reference to The Godfather with Gru finding the stuffed unicorn's head in his bed and freaking out.
Gru speaking to Dr. Nefario over the video intercom, and acting as if everything is normal... despite having Kyle hanging off his arm, and eating his muffin, having sunk his teeth into it.
When Gru brings the girls home; Agnes spots Kyle, shrieks "Ooh, fluffy doggie!" and runs up to give him a hug — whereupon he yelps in fright and runs away. Priceless.
One of the recently-released clips should qualify on its own. Someone has come to Gru's door to set him up with a date, and Gru really doesn't want to go...Problem is, he's letting Agnes make excuses for him.
Gru:*makes 'zip it' movement*
Agnes: "He's...putting on lipstick!"
Gru:*quickly swings his arms around, trying to convey 'stop'*
