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The graduate experience is a critical time for development of academic faculty, but often there is little preparation for teaching during the graduate career. Teaching self-efficacy, an instructor’s belief in his or her ability to teach students in a specific context, can help to predict teaching behavior and student achievement, and can be used as a measure of graduate students’ development as instructors. An instrument measuring teaching self-efficacy of science, technology, engineering, and mathematics (STEM) graduate teaching assistants (GTAs) was developed from a general university faculty teaching instrument to the specific teaching context of STEM GTAs. Construct and face validity, measurement reliability, and factor structure of the instrument were determined from survey data of 253 STEM GTAs at six universities. STEM GTA teaching self-efficacy correlated to various measures of GTA professional development and teaching experience. Implications and applications for faculty involved in GTA professional development, supervision, and research are discussed.