Mitochondria host the machinery for the tricarboxylic acid (TCA) cycle and electron transport chain (ETC), which generate adenosine triphosphate (ATP) to maintain energy homeostasis. Glucose, fatty acids, and amino acids are the major energy substrates fueling mitochondrial respiration in most somatic cells. Evidence shows that different cell types may have a distinct preference for certain substrates. However, substrate utilization by various cells in the skeleton has not been studied in detail. Moreover, as cellular metabolism is attuned to physiological and pathophysiological changes, direct assessments of substrate dependence in skeletal cells may provide important insights into the pathogenesis of bone diseases. The following protocol is based on the principle of carbon dioxide release from substrate molecules following oxidative phosphorylation. By using substrates containing radioactively labeled carbon atoms (14C), the method provides a sensitive and easy-to-use assay for the rate of substrate oxidation in cell culture. A case study with primary calvarial preosteoblasts versus bone marrow-derived macrophages (BMMs) demonstrates different utilization of the main substrates between the two cell types.