The isolation of high-purity cellular biomacromolecules and sub-cellular organelles is an essential aspect to mass spectrometry based studies. Mitochondria are sub-cellular organelles that perform a central role in cellular energy production. Mitochondria are of great interest due to their potential to generate reactive oxygen species (ROS) and susceptibility to oxidative damage and subsequent functional impairment. Current methods of mitochondria isolation are optimized for respiratory-based studies that favor viability. Whereas, proteomic and lipidomics studies of mitochondria require procedures that optimize for purity and enrichment. We describe a procedure derived from previously established methods for the isolation of mitochondria, nuclear and cytosolic fractions from a neurological tissue sample. In addition to the isolation being of significant purity for mass spectral based '-omics' analysis, mitochondrial yields were routinely 500 μg per tissue wet weight, allowing multiple studies to be conducted from a single isolation procedure.