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The oxygen consumption rate of tumor cells affects tumor oxygenation and response to therapies. Highly sensitive methods for determining cellular oxygen consumption are, therefore, needed to identify treatments that can modulate this parameter. We compared the performances of three different methods for measuring cellular oxygen consumption: electron paramagnetic resonance (EPR) oximetry, the Clark electrode, and the MitoXpress fluorescent assay. To compare the assays, we used K562 cells in the presence of rotenone and hydrocortisone, compounds that are known to inhibit the mitochondrial electron transport chain to different extents. The EPR method was the only one that could identify both rotenone and hydrocortisone as inhibitors of tumor cell oxygen consumption. The Clark electrode and the fluorescence assay demonstrated a significant decrease in cellular oxygen consumption after …