Neurons critically depend on mitochondrial function to establish membrane excitability and to execute the complex processes of neurotransmission and plasticity. Although mitochondria are central for various cellular processes important for neuronal function that include ATP production, intracellular calcium signaling, and generation of reactive oxygen species, our understanding of the roles of mitochondria in neurons and intact brain tissue are limited. Proteins carry out an array of mitochondrial functions, with structural, transporter, hormone, enzyme, and receptor protein classes working in complexes and pathways to serve these critical functions. Knowledge of the protein composition of mitochondria and how it changes under different conditions will yield important information to further our insight into mitochondrial processes. Here we describe the implementation of mass spectrometry (MS)-based proteomics to the study of neuronal mitochondria, with emphasis on quantitative approaches. This chapter describes the isolation of mitochondria from primary neuronal cultures and brain tissue nerve terminals, sample preparation for liquid chromatography dual MS (LC-MS/MS), the LC-MS/MS procedure, and the subsequent data processing steps to compare global mitochondrial proteomic alterations. Identifying mitochondrial proteins that are altered within neurons during development and disease is of central importance for understanding the multifaceted roles of mitochondria.