Mitochondria play a key role in cellular metabolism. Analyses of the genome, the proteome, metabolic, physiological, and biochemical functions of mitochondria frequently require the isolation of intact and functional mitochondria from various plant tissues with sufficient yield. For this purpose, we generated a transgenic Arabidopsis thaliana (Arabidopsis) line which presents a triple hemagglutinin tag on the surface of the outer mitochondrial membrane. The affinity tag enables immunocapture of the organelles in a single step. This chapter gives detailed instructions on how to generate transgenic Arabidopsis lines harboring a ubiquitously expressed 3xHA-sGFP-TOM5 mitochondrial fusion protein that is targeted to the outer mitochondrial membrane and enables purification of the organelles in a single step.