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Glaser was born in Cinncinati, Ohio. She has German and Irish ancestry.Glaser mostly grew up in St. Louis, Missouri.
Glaser started performing stand-up at the age of 18. Less than two years later, she was a semi-finalist on Last Comic Standing. Since then, Glaser has performed stand-up on The Tonight show with Jay Leno,Conan, and two seasons of Last Comic Standing. From 2011-2014, Glaser hosted a podcast with comedian Sara Schaefer called You Had To Be There.
In March 2014, Glaser appeared on Giants Bomb's life advice podcast, Danswers, talking about subjects such as her experience with co-host Dan Ryckert at college.
Glaser co-hosted the podcast We Know Nothing with comedian Phil Hanley and roommate/musician Anya Marina. We Know Nothing is a humorous relationship-based podcast that takes calls from listeners and attempts to give them love advice. In 2015, she left the podcast after she determined that it was interfering with her personal relationships.
Nikki&Sara Live, a weekly talk show television series hosted by Glaser and Schaefer, premiered on January 29, 2013, on MTV.The show was canceled on November 22, 2013, after two seasons.
Glaser has also appeared on MTV shows such as Money from Strangers,Awkward, After Show,You're Welcome, and Failosophy.
On June 2, 2015, Comedy Central green lit a new sex-themed talk show hosted by Glaser, titled Not Safe With Nikki Glaser. Ten episodes have been ordered, and the show is scheduled to premiere on February 9, 2016