SummaryDetection of endogenous circadian rhythms in clock gene mRNA expression requires that mice be sacrificed at regular time intervals over one or more days. This protocol uses culture tissue slices obtained from a single mouse to collect time-course samples. We describe the procedure from preparation of lung slices to rhythmicity analysis of mRNA expression, including details to create handmade culture inserts. This protocol is useful for many mammalian biological clock researchers because it allows a decrease in animal sacrifice.For complete details on the use and execution of this protocol, please refer to Matsumura et al. (2022).1