From 1989 to 1993, Whitson worked as a research biochemist in the Biomedical Operations and Research Branch at NASA-JSC. From 1991 to 1993, she served as technical monitor of the Biochemistry Research Laboratories in the Biomedical Operations and Research Branch. From 1991 through 1992, she was the payload element developer for Bone Cell Research Experiment (E10) aboard SL-J (STS-47), and was a member of the US-USSR Joint Working Group in Space Medicine and Biology. In 1992, she was named the project scientist of the Shuttle-Mir Program (STS-60, STS-63, STS-71, Mir 18, Mir 19), and served in this capacity until the conclusion of the Phase 1A Program in 1995. From 1993 through 1996, Whitson held the additional responsibilities of the deputy division chief of the Medical Sciences Division at NASA-JSC. From 1995–1996, she served as co-chair of the U.S.-Russian Mission Science Working Group.

In April 1996, Whitson was selected as an astronaut candidate and started training in August 1996. Upon completing the two years of training and evaluation, she was assigned technical duties in the Astronaut Office Operations Planning Branch, and served as the lead for the Crew Test Support Team in Russia from 1998 to 1999. In June 2003, Whitson served as the commander of the NEEMO 5 mission aboard the Aquarius underwater laboratory, living and working underwater for fourteen days.^[18] From November 2003 to March 2005, she served as deputy chief of the Astronaut Office. From March 2005 to November 2005, she served as chief of the Station Operations Branch, Astronaut Office. Whitson trained as the backup ISS commander for Expedition 14 from November 2005 through September 2006, and as the ISS commander for Expedition 16, launched October 2007, on the Soyuz TMA-11. During Expedition 16 she surpassed Sunita Williams for woman with the most spacewalks.