Science, technology, engineering, and mathematics education reform efforts have called for widespread adoption of evidence-based teaching in which faculty members attend to student outcomes through assessment practice. Awareness about the importance of assessment has illuminated the need to understand what faculty members know and how they engage with assessment knowledge and practice. The Faculty Self-Reported Assessment Survey (FRAS) is a new instrument for evaluating science faculty assessment knowledge and experience. Instrument validation was composed of two distinct studies: an empirical evaluation of the psychometric properties of the FRAS and a comparative known-groups validation to explore the ability of the FRAS to differentiate levels of faculty assessment experience. The FRAS was found to be highly reliable (α = 0.96). The dimensionality of the instrument enabled distinction of assessment knowledge into categories of program design, instrumentation, and validation. In the known-groups validation, the FRAS distinguished between faculty groups with differing levels of assessment experience. Faculty members with formal assessment experience self-reported higher levels of familiarity with assessment terms, higher frequencies of assessment activity, increased confidence in conducting assessment, and more positive attitudes toward assessment than faculty members who were novices in assessment. These results suggest that the FRAS can reliably and validly differentiate levels of expertise in faculty knowledge of assessment.