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A key challenge for teachers is the evaluation of teaching and learning situations in the classroom. Video-based instruments are considered as effective tools for measuring teachers’ skills for evaluating classroom situations. This article reports the development and validation of a video-based assessment tool for measuring biology teachers’ professional vision. Analyses of interviews using think-aloud protocols with in-service biology teachers showed that they perceived the staged videos in the video-based assessment tool as authentic. Furthermore, they identified biology-specific dimensions of instructional quality in the videos, and used aspects of professional vision while reasoning about the identified situations. Thus, results indicate the suitability of the video-based assessment tool for capturing professional vision with the potential to be used in further interventions within teacher education programs.