An analytical technique for the detection of permeation of a fully ionized analyte across a lipophilic membrane is reported. The system, which is comprised of two aqueous compartments (donor and acceptor) separated by a supported liquid membrane, is based on the parallel artificial membrane permeation assay (PAMPA), widely used in the drug discovery process to estimate permeability in vivo. The in situ spectroelectrochemical method developed here employs mechanical stirring of the solution phases on either side of the membrane, external polarization of the membrane, and in situ detection of the analyte via UV-vis spectrophotometry. The flux of the crystal violet cation across the membrane is simultaneously measured via UV-vis spectrophotometry and voltammetry/chronoamperometry as a function of applied potential. The relative contribution of two permeation modes, i.e., that due to naked ions and ion-pairs, is thereby quantified. The open circuit potential difference between the two aqueous compartments and the cyclic voltammetric response are also recorded as a function of time and compared with the predicted values. © 2012 American Chemical Society.