Jhonen starts as a cartoonist in the high school's student newspaper, publishing comic strips featuring a character: Jonny, the little homicidal maniac. After graduating in 1992 he began film classes at De Anza College, but he dropped out to pursue a career as a professional cartoonist.
Carpe Noctem Magazine start publishing one-page strips featuring Jonny the homicidal maniac, and in 1995 Slave Labor Graphics began publishing a comic book of the same character. The Johnny comic ran for seven issues. In 1997 Vasquez published Squee, a supporting character from Jonny the homicidal maniac, in his own 4 issues series, and soon came out I Feel Sick, featuring Devi, “a girl who tries to maintain her sanity” (and another character from Jonny).
With the fame acquired as an independent creator, Nickelodeon decided to hire him to develop a television show for their older demographic, and that is how came out Invader Zim, originally pitched in 1999. The show premiered in 2001 and was acclaimed by critics, won three awards (including one Emmy Award and one Annie Award), and other seven nominations. However, the show was canceled in 2002. Since then, despite its early cancellation, it has increased the popularity of the show until being considered a cult classic.
After that Slave Labor has published other Jhonen works, and he had several collaborations, including his participation in two issues of Marvel Comic's Strange Tales, and as a writer in DC. He has also collaborated in designing characters for Disney XD among others, and was a director of two music videos.
In 2015, Oni Press began publishing a comic book series of Invader Zim, in collaboration with Jhonen Vasquez and Nickelodeon, as the continuation of the TV series. In 2017 Nickelodeon announced a 66-minute television film based on the series, with the collaboration of Jhonen Vasquez as executive producer.