The ability to detect and identify mitochondrial proteins that are sensitive to oxidative modification and inactivation by reactive species is important in understanding the molecular mechanisms responsible for mitochondrial dysfunction and tissue injury. In particular, cysteine residues play critical roles in maintaining the functional and structural integrity of numerous proteins in the mitochondrion and throughout the cell. To define changes in mitochondrial protein thiol status, proteomic approaches have been developed in which unmodified, reduced thiols (i.e., R–SH or thiolate species R–S−) are tagged with thiol-labeling reagents that can be visualized following gel electrophoresis and immunoblotting techniques. Herein, we describe the use of one thiol-labeling approach in combination with blue native gel electrophoresis (BN-PAGE) to detect reactive thiol groups within mitochondrial proteins including those of the oxidative phosphorylation (OxPhos) system. Labeling or “tagging” of protein thiol groups in combination with various gel electrophoresis and proteomics techniques is a valuable way to measure alterations in cellular or organelle thiol proteomes in response to drug treatment, disease state, or metabolic/oxidative stress.