Using a novel permeation measurement methodology based on a liposomal assay, the permeation enhancement or suppression during co-permeation of two substances has been directly investigated for the first time. As a methodology validation after the selection of an appropriate temperature, two agents with known permeation enhancement properties due to membrane disruption were studied (ethanol and oleic acid). In the case of ethanol addition, a stepwise release of the permeant (CF) was observed. This was due to the extraction of lipids from the membrane by ethanol and the loss of membrane integrity in the affected the liposomes from which CF could leak out. Oleic acid worked on a different principle, which, due to its incorporation into the membrane, caused gradual permeation of CF even at 30 °C, i.e., well below the phase transition of the original membrane. A mathematical model of permeation enables the quantitative evaluation of permeability and the membrane partitioning coefficient of the permeant.