DeStefano's first credit in the comics industry was as the creator of the character Captain Electron in Adventure Comics #479 (March 1981), and then the characters Zeep and Thumbelina in the "Dial H for Hero" story in Adventure Comics #483 (July 1981).[3] His first professionally published comics artwork was a one-page parody of the "I…Vampire" feature in House of Mystery #306 (July 1982).[4] DeStefano wrote and drew "The Mini (Mis)Adventures of Nick O. Tyme" in New Talent Showcase #6–12 (June–December 1984). He then teamed with writer Bob Rozakis to create 'Mazing Man, a character introduced in an eponymous series beginning in January 1986.[5] After the cancellation of that series, the Rozakis-DeStefano team created Hero Hotline in Action Comics Weekly #637 (January 1989).[6] After a four-issue run as a back-up feature, Hero Hotline received its own six-issue limited series.[7] In the 1990s, DeStefano was a frequent contributor to The Big Book Of series published by Paradox Press. His other comics work includes credits at Dark Horse Comics, Disney Comics, Fantagraphics Books, and Marvel Comics.[4][8] As an animator, DeStefano has worked on such series as The Ren & Stimpy Show and Futurama.[2][9] He inked the Cage limited series drawn by Genndy Tartakovsky in 2016–2017.[10] In 2020, DeStefano won the Outstanding Individual Achievement in Animation Emmy for Character Design on the Genndy Tartakovsky series Primal.[11] In 2024, he contributed art to the Alex Hirsch book The Book of Bill.[12]