Mitochondrial dysfunction and mutations in mitochondrial DNA (mtDNA) have been frequently reported in cancer, neurodegenerative diseases, diabetes, and aging syndromes. The mitochondrion genome (16.5 Kb) codes only for a small fraction (estimated to be 1%) of the proteins housed within this organelle. The other proteins are encoded by the nuclear DNA (nDNA) and transported into the mitochondria. The identification of mitochondrial proteins that are aberrantly expressed in cancer cells and other diseases is now possible through recent developments in proteomic and bioinformatic technologies. These developments set the stage for a comprehensive organelle-based proteomic approach for the identification of new markers for the early detection, risk assessment, and diagnosis of cancer, and other diseases and for the identification of new targets for therapeutic prevention and intervention.