Mitochondrial dysfunctions decisively contribute to the progression of human diseases, implying that functional tests of isolated mitochondria may furnish conclusive information for diagnosis and therapy. Classical mitochondrial isolation methods, however, lack precisely adjustable settings for cell rupture, which is the most critical step in this procedure, and this complicates subsequent analyses. Here, we present an efficient method to isolate functionally active, intact mitochondria from cultured or primary cells and minute tissue samples in a rapid, highly reproducible manner.