One major problem concerning the analysis of mitochondria is the heterogeneity of mitochondrial appearance, especially under pathological conditions. We show here the use of zone-electrophoresis in a free-flow device (ZE-FFE) as an analytical sensor to discriminate between different yeast mitochondrial populations. Impairment of the organelles’ structural properties by hyper-osmotic stress resulted in broad separation profiles. Conversely, untreated mitochondria gave rise to homogeneous populations reflected by sharp separation profiles. Yeast mitochondria with altered respiratory activity, accompanied by a different outer membrane proteome composition, could be discriminated based on electrophoretic deflection. Proteolysis of the mitochondrial surface proteome and the deletion of a single major protein species of the mitochondrial outer membrane altered the ZE-FFE deflection of these organelles. In order to demonstrate the usefulness of ZE-FFE for the analysis of mitochondria associated with pathological processes, we analyzed mitochondrial fractions from an apoptotic yeast strain. The cdc48S565G strain carries a mutation in the CDC48 gene that is an essential participant in the ER-associated protein degradation pathway. Mutant cells accumulate polyubiquitinated proteins in microsomal and mitochondrial extracts. Subsequent ZE-FFE characterization could distinguish a mitochondrial subfraction specifically enriched with polyubiquitinated proteins from the majority of non-affected mitochondria. This result demonstrates that ZE-FFE may give important information on the specific properties of subpopulations of a mitochondrial preparation allowing a further detailed functional analysis.