This study presents the systematic development, validation, and use of a new instrument for measuring student interest in science and technology. The Student Interest in Technology and Science (SITS) survey is composed of 5 sub-sections assessing the following dimensions: interest in learning science, using technology to learn science, science careers, technology careers, and attitudes toward biotechnology. Our development process included review of existing instrumentation, pilot testing, and expert panel review. The resulting instrument was administered before and after implementation of a biotechnology intervention which used a computer-based game to engage learners in the use of biotechnology to address a societal issue. We employed item response theory (IRT) to explore instrument validity and precision. Results of the psychometric analyses suggest that the SITS survey has a well-defined structure and meets IRT assumptions. Difficulty and discrimination parameters as well as reliability analyses indicate that SITS items provide useful measures of student interest. Finally, we use the SITS to explore the extent to which the intervention used in this study supports changes in student interest and association between students’ interest and related content knowledge. Implications for the future use of this instrument are discussed.