Mitochondria are responsible for a number of major biochemical processes in plant cells including oxidative phosphorylation and photorespiration. Traditionally their primary role has been viewed as the oxidation of organic acids via the tricarboxylic acid cycle and the synthesis of ATP coupled to the transfer of electrons to O2. More recently its role in the synthesis of many metabolites such as amino acids, lipids, and vitamins has been revealed. They also contain large number of transporters including members of the mitochondrial carrier substrate family (MCSF) that allow the exchange of metabolites with the cytosol. Mitochondria also contain their own genome and actively transcribe and translate a set of proteins that are coordinated with proteins encoded by the nuclear genome to produce large multisubunit enzymes. To reveal the full diversity of metabolism carried out by mitochondria significant efforts have sought to uncover the protein profile of mitochondria from both crops and model plants. Successful proteomic analysis depends on the preparation of high-quality isolated mitochondria, coupled to high-resolution proteomic techniques for identification, quantitation, and assessment of the degree of contamination by other organelles and cellular compartments. Here we outline a mitochondrial isolation protocol that can be applied to a range of plant tissues, and detail methods of assessing the quality and purity of the resultant sample, including calculations of respiratory control ratio, marker enzyme assays, differential in-gel electrophoresis, and quantitative gel-free mass spectrometry.