Updated publication reference for PubMed record(s): 31851916. Mitochondrial DNA mutations progressively compromise the respiratory chain of skeletal muscle, resulting in a mosaic of metabolically healthy and defective fibers. This heterogeneity is the best diagnostic signpost of mitochondrial disorders. However, the single fiber investigation of its molecular basis has been beyond the capability of large-scale technologies so far. We here use a novel approach to single muscle fibers proteomics, based on laser capture microdissection to excise thin sections of individual muscle fibers from frozen biopsies. Combined with a highly sensitive mass spectrometry-based proteomics workflow, this technique allowed us to analyze healthy and defective muscle fibers from patients with mitochondrial disorders.