Dissatisfied with the Seattle scene, Marsters headed to Los Angeles in the mid-90s. He landed the role of 200-year-old vampire William the Bloody (nicknamed 'Spike' after his favored weapon) on "Buffy the Vampire Slayer" in 1997. A former pupil of David Boreanaz's Angel, Spike wasn't burdened by the conscience that plagued his lovestruck mentor, and was a welcome villainous addition to the "Buffy" stable of supernatural characters, offering comic relief to the sometimes overearnest series. Marsters' skillful North London accent and wildly acerbic delivery made lines such as "I love syphilis more than I love you" (delivered to Juliet Landau's Drucilla, Spike's girlfriend) all the more enjoyable. Spike's popularity won the actor a regular spot in the cast in 1999, and in order to counteract his unabashed evil, writers hindered Spike's murderous glee by adding a plot point that saw the bloodsucker altered by a mysterious group called the Initiative. The result was that whenever Spike did harm, he feels unbearable physical pain. As he became an integral part of the otherworldly teen clan, Spike ironically began to fall for the eponymous slayer (Sarah Michelle Gellar).

As "Buffy" raised his profile, Marsters appeared on the big screen in the 1999 horror thriller "The House on Haunted Hill". He didn't abandon stage work despite his TV stardom, starring and contributing to the score of the ambitious drama "The Why" (2000), produced by the Blank Theater Company. Marsters also took his singing and guitar playing talents to the stage with a series of acoustic club performances in 2000.