Boosting students' disciplinary interest has long been considered an important mechanism to increase student success and retention in STEM education. Yet, interest is a complex construct and can mean different things to different people, and many of the existing interest questionnaires do not identify a specific theoretical framework underlying their items. To demonstrate that curricular interventions targeting students' interest are effective, educators need a theoretically based instrument to measure interest. The aim of this study was to develop an instrument measuring undergraduate students' interest in the discipline of biology and collect initial validity evidence supporting the proposed use. The instrument structure is based on Hidi and Renninger's (Educational Psychologist 41:111-127, 2006) conceptualization of individual interest, and the intended use is to evaluate changes in the biology interests of the US undergraduate students pursuing STEM degrees. To provide evidence of validity, the instrument was completed by 446 biology majors and 489 non-biology majors at two R1 universities. Exploratory and confirmatory factor analyses were applied to evaluate the internal structure of the instrument.The final three-factor instrument supported by these analyses includes 6 items representing positive feelings towards biology, 5 items representing personal value of biology, and 8 items representing reengagement in biology-related activities. Measurement invariance across biology and non-biology majors was established and subsequent comparisons of these populations demonstrated that biology majors report significantly higher positive feelings, personal value, and reengagement in biology-related activities compared to non-biology majors.The study findings support the use of the instrument to gain a broad understanding of students' individual interest in biology. With minor adaptions, the instrument could also be evaluated for use in other STEM disciplines and for use by other populations.