Graduate teaching assistants (TAs) may identify with multiple roles during their introductory biology teaching experiences. This study developed an instrument that measures TA role identity in introductory biology. Initial development and content validation of the Biology Teaching Assistant Role Identity Questionnaire (BTARIQ) occurred in four phases. The first phase included the specification of instrument purpose and descriptions of the conceptual definitions. The second focused on item generation mapped to the constructs and dimensions based in the literature. An expert review panel validated the instrument content for the constructs, and a draft of the instrument was developed based on this review. The third consisted of a pilot to a small group of the target population using the draft instrument, and the instrument was further revised based on the results. In the final phase, an exploratory Rasch analysis was conducted with a field test sample to examine dimensionality, model fit, person and item separation and reliability, and rating scale category functioning. Findings suggest some evidence to support the interpretation and use of scores from the BTARIQ.