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Summary: Non-invasive measurement of circadian rhythms is important for longitudinal assays of the rhythmic swimming behavior of the turquoise killifish (Nothobranchius furzeri). Here, we introduce a custom-built, video-based system for non-invasive circadian rhythm measurement. We describe imaging tank setup, video recording and editing, and fish movement tracking. We then detail circadian rhythm analysis. This protocol can be used for repetitive and longitudinal analysis of circadian rhythms in the same fish with minimal stress and can be applied to other fish species.For complete details on the use and execution of this protocol, please refer to Lee et al..1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.