Proteomic analysis of organelles or specific groups of proteins is a powerful strategy for the discovery of proteins that are involved in specific cellular functions or disease. Targeted enrichment of specific groups of proteins or subcellular organelles reduces the complexity of samples and simplifies such approaches. As they are the organelles in animal and plant cells in which oxidative phosphorylation takes place, mitochondria are the site of energy production in eukaryotic cells. In tissues that require large amounts of energy (e.g., skeletal muscle, kidney, or the pancreas), there are noticeably more mitochondria than in tissues with lower energy requirements. Mitochondria are compartmentalized by an outer and highly folded inner membrane. The inner membrane encloses the mitochondrial matrix, which contains the enzymes that take part in the Krebs respiratory cycle. The molecules that perform electron transfer and ATP synthesis are located on folds (cristae) on the inner surface of the inner membrane. Due to their fundamental importance in biochemistry, mitochondria are a subject of intense research. Mitochondrial dysfunction can lead to serious and debilitating medical conditions, such as cancer, infertility, diabetes, heart diseases, blindness, deafness, kidney disease, liver disease, stroke, and migraine. Mitochondrial dysfunction is also involved in aging and neurodegenerative diseases such as Parkinson’s and Alzheimer’s disease.