A pathological increase of the permeability of the mitochondrial membranes may culminate in the irreversible rupture of the mitochondrial outer membrane. Such a permeability transition is lethal because it results in the release of death-inducing molecules from mitochondria and/or metabolic failure. Current methods to assess this outer membrane damage are mostly indirect or scarcely representative of the overall mitochondrial population. Here we present an analytical and preparative approach using free flow electrophoresis to directly distinguish rat liver mitochondria that have undergone the permeability transition from unaffected organelles or from organelles that are damaged to a minor degree. Mitochondrial populations, which considerably differ in outer membrane integrity or cytochrome c content, were separated by this means. We further show that the relative abundance of each population depends on the dose of the permeability transition inducer and the duration of the treatment time. Finally, we have employed this approach to investigate the impairment of mitochondria that were isolated from livers subjected to ischemia/reperfusion damage.