Updated project metadata. Photosynthesis in plant cells would not be possible without the supportive role of mitochondria. However, isolating mitochondria for physiological and biochemical analyses from plant cells is a lengthy and tedious process. Established isolation protocols require multiple centrifugation steps and substantial amounts of starting material. To overcome these limitations, we tagged mitochondria in plant cells with a triple haemagglutinin-tag for rapid purification by a single affinity purification step. This protocol yields highly purified mitochondria from 1 g of Arabidopsis seedlings that are suitable for enzyme activity measurements and delivers sufficient amounts of mitochondrial proteins for deep proteomic profiling. We demonstrate that the method can be applied to proteomic analysis of an Arabidopsis mutant deficient in the mitochondrial glutamate transporter À bout de souffle (BOU) and we identify 27 differentially expressed proteins in the bou mutant. Our work also sets the stage for the development of advanced isolation protocols for mitochondria from distinct cell types.