For many metabolites, the major barrier between cytosol and mitochondrial matrix is the inner membrane of mitochondria, the site of the respiratory electron transport chain. In consequence, it houses numerous transporters which facilitate the controlled exchange of metabolites, ions, and even proteins between these cellular compartments. While their import into the organelle can be studied with isolated mitochondria or mitoplasts, the analysis of their export from the matrix into the intermembrane space or even the cytosol demands for more sophisticated approaches. Among those, inside-out inner membrane vesicles are particularly useful, since they allow the direct presentation of the potential export substrates to the membrane without prior import into the organelle. Here we present a protocol for the isolation of such inside-out vesicles of the inner membrane of plant mitochondria based on repeated freeze/thaw-cycles of freshly prepared mitoplasts. Electron microscopy and Western analysis could show that the majority of the vesicles have single envelope membranes in an inside-out topology. The vesicles are furthermore physiologically active, as demonstrated by assays measuring the enzymatic activities of Complex I (NADH dehydrogenase), Complex V (ATP synthase) and the mitochondrial processing peptidase (MPP) associated with Complex III. Hence, the method presented here provides a good basis for further studies of the inner mitochondrial membrane and mitochondrial export processes.