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This chapter focuses on the biochemical assays of the respiratory chain (RC) complex activity. It presents the preparation of mitochondrial fractions from tissues and cultured cells for RC enzymology; the measurement of activity of the individual complexes I, II, III, IV, V, the mitochondrial matrix marker enzyme citrate synthase; and the combined activity of complexes II + III. RC enzyme activities are frequently expressed relative to its activity of citrate synthase. Such ratios are more robust than absolute activities because of the variability inherent in cell culture conditions, such as passage number and the degree of confluence, and the proliferation of mitochondria seen in tissues of many patients with mitochondrial disease. The effects of postmortem delay on RC enzymes from liver samples frozen at varying times after death were analyzed to assess the stability of RC enzyme activities postmortem …