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As implementation of green chemistry into university-level courses increases, it is vital that educators have a tool to rapidly measure student knowledge of green chemistry principles. We report the development of the Assessment of Student Knowledge of Green Chemistry Principles (ASK-GCP) and evaluation of its sensitivity and effectiveness for measuring student knowledge of green chemistry. The 24-item true–false instrument was given to a total of 448 students to gather data on the reliability, validity, and sensitivity. The instrument proved to be sensitive for distinguishing known groups with various levels of green chemistry knowledge and instructional exposure. The instrument was able to detect gains in green chemistry knowledge in pre- and post- conditions. Psychometric analysis revealed that the item difficulty range matches the sample ability range. The findings verified that the ASK-GCP is an efficient and accurate instrument to measure student knowledge of green chemistry principles.