Made even better in the commentary when the directors and writers discuss how the screenwriter voiced the girly scream, and he was a man. They tried getting a real woman to scream, a child to scream, but decided to keep the screenwriter's scream, because "no-one can scream like a little girl like he can."
President Buffoon in Monsters vs. Aliens shrieks like a frightened girl the first time he sees Ginormica. Especially amusing since the previous four identical girly screams were from an actual girl. The scene is even funnier when you consider that Ginormica was the only one of the monsters that scared him. Shows that a powerful, unstoppable woman is what all men fear most — even the President!
In one of the trailers for the CGI film Chicken Little, the title character's father warns him that in a few seconds he's going to scream like a little girl as they try to avoid the meteorites being hurled upon them. Sadly, this never happensin the actual film.
Spike:Any last requests? Roddy: Yes. Could you fly, quite suddenly, off the boat, screaming like a girl? Spike: What? (is jerked off the boat by the cord he's attached to) AAAAAAAAAAH!
In The Incredibles, Syndrome does this when Jack-Jack goes demon-baby, pulls out his hair. Also, just as he's about to die. Foreshadowed when Syndrome is taunting Mr. Incredible at his island base.
In The Road to El Dorado, upon both Miguel and Tulio (pictured on this trope's main page) seeing the giant stone Jaguar activated by Tzekel-Kan, they perform a seldom seen double girly scream. Even better, if you listen carefully enough, you can tell that there are two distinct screams and, more importantly, which scream belongs to who (Miguel's scream is the higher pitched one).
Roxanne points out that Megamind does a pretty good example.
Lampshaded in Resident Evil: Degeneration when Claire and the other survivors are holed up in the small office they hear a high-pitched scream and Claire decides to investigate. A few scenes later she comes back with an unknown man and people ask where the "girl" is.
In Open Season, Ian, Manliest of Mule Deer Stags, is galloping gracefully with his herd across the river when the floodwater comes down. Mid-leap he turns to look at the rushing water and gives a girly scream just as it hits.
Ratchet from Robots screams like this a lot whenever he's in trouble.
In Aladdin, the Genie screams like a girl many times. For example, when Aladdin rubs the lamp for the first time and lets him out. And when Iago introduces Kassim to the Genie as the King of Thieves, before Aladdin introducing Kassim to the Genie as his father.
In Big Hero 6, Wasabi, the largest and toughest-looking of the team, screams like a little girl when he's nearly crushed by a shipping container thrown by Yokai.