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She first broke into comedy as a featured player in the Chicago-based improv comedy group The Second City. She then joined SNL as a writer, later becoming head writer and a performer, known for her position as co-anchor in the Weekend Update segment. In 2004, she adapted the screenplay Mean Girls in which she also co-starred.

After leaving SNL in 2006, she created the television series 30 Rock, a situation comedy loosely based on her experiences at SNL. In the series, she portrays the head writer of a fictional sketch comedy series. In 2008, she starred in the comedy film Baby Mama, alongside former SNL co-star Amy Poehler. She next appeared alongside Steve Carell in the 2010 comedy film Date Night and with Will Ferrell in the animated film Megamind.

In 2008, the Associated Press (AP) gave her the AP Entertainer of the Year award for her satirical portrayal of Republican vice presidential candidate Sarah Palin in a guest appearance on SNL. In 2010, she was the recipient of the Mark Twain Prize for American Humor, the youngest-ever winner of the award.

On January 13, 2013, she hosted the Golden Globe Awards with her long-time friend and fellow comedian, Amy Poehler. Their performance was critically acclaimed. The duo hosted again the following year to similar acclaim, generating the highest ratings for the annual ceremony in ten years.