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Summary: Mitochondrial respirometry allows for the comprehensive study of oxygen consumption within the electron transport system in tissues. However, limited techniques exist for analyzing frozen or biobanked intestinal tissues. Here, we present a protocol to evaluate the respiratory function of mitochondria in colonic tissues after cryopreservation at −80°C. We describe steps for rat dissection, respirometry calibration, and tissue preparation. We then detail measurement of oxygen respiration and protein concentration. This protocol facilitates the retrospective analysis of mitochondrial respiration in frozen tissue. : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.