Mitochondria carry out a variety of important processes in plants. Their major role is the synthesis of ATP through the coupling of a membrane potential to the transfer of electrons from NADH to O₂ via the electron transport chain. The NADH is generated by the oxidation of organic acids via the tricarboxylic acid cycle. However, mitochondria also perform many important secondary functions such as synthesis of nucleotides, amino acids, lipids, and vitamins. Mitochondria contain their own genome and undertake transcription and translation by some unique mechanisms; they actively import proteins and metabolites from the cytosol, are involved in programmed cell death processes in plants, and respond to cellular stress conditions. To understand the extent and mechanisms of mitochondrial functions in plants and the way in which their functions are perceived by the nucleus requires detailed information about the protein components of these organelles. Isolation of mitochondria to identify their proteomes and the changes in these proteomes during development and environmental stresses is growing area of research. In this chapter we provide a useful method for the isolation of mitochondria from plant cell culture using a gentle method of cell disruption based on protoplasts isolation that provides relatively high mitochondrial yields.