He began his voice over career on The Smurfs animated television show as an extra. A few years later, his career launched into acting on more famous cartoons, such as G.I. Joe and The Snorks. He had an impressive turn as the villain Jesse Blue in the series Saber Rider and the Star Sheriffs. The character has a bit of a cult following, due in part to Rob's excellent portrayal. In 1987, Rob was asked to voice one of his most prominent roles, as Raphael in the 1987 Teenage Mutant Ninja Turtles television series. He also voiced some minor roles in that television series. He also played a role in the TV series Cheers as the show's announcer.

With a plethora of voice acting roles under his belt, it was only a matter of time that he would basically be granted his own show. In 1993, he voiced Antoine D'Coolette in ABCs series Sonic the Hedgehog, and Yakko Warner of Animaniacs, an animated television show that revolved around Yakko and his siblings. This eventually led to another series where he led as Pinky from Pinky and the Brain, a show which won him several Annie Awards and a Daytime Emmy in 1999. Also in 1993, he starred as the title character in two animated series: Mighty Max and The Mask. He also posed as the title character's sidekick, Arthur, an insecure accountant in a moth costume (wings included), in the superhero series The Tick.

He recently took on the role of Experiment 625 in Lilo & Stitch: The Series. Rob also currently does the roles of Jack Fenton and the Box Ghost of Danny Phantom, Carl Wheezer and Butch the bully of Jimmy Neutron and the series, and Gordon on the Nickelodeon cartoon Catscratch. In the new world of Warner Bros. Animation, he also provides the voice of the hero, Rev Runner of the new show Loonatics Unleashed and the voice of Coconut Fred in Coconut Fred's Fruit Salad Island. He also voiced The Source in the Teen Titans episode "Employee of the Month". In total, Rob Paulsen has been the voice of over 200 different animated characters. Chances are, if you've watched cartoons you've heard Rob Paulsen's voice.

Paulsen continues to do many voice roles, now extending into video games such as Doom 3 and the Metal Gear Solid franchise. He played the voice of Morte, a floating, talking skull, in Planescape: Torment. He voiced the Cyborg Ninja in Metal Gear Solid: The Twin Snakes, an enhanced remake of the original Metal Gear Solid, and he also voiced Crash Bandicoot in many of the later games for Nintendo GameCube, Sony PlayStation 2, and Microsoft Xbox platforms. He also provides the voice for Bonker the Clown in Clay Fighter 63 1/3. More notably, he voiced Tobli and Lian Ronso in the English version of Square Enix's Final Fantasy X-2, the sequel to Final Fantasy X. He has also played the voice of Bubsy in the game series of the same name (specifically, the second game; Brian Silva was the voice of Bubsy in the first) and an art teacher in an episode of The Boondocks. In the early 2000s, Paulsen provided the voice of "Mr. Opportunity" in a series of radio and TV commercials for Honda.

And even though it was an extremely minor role, Paulsen also has done the voice for the Greek Soldiers in God of War.