Purpose: The objective of this study was to develop a non-invasive technique to assess the interaction between chemical and electrical modes of percutaneous penetration enhancement in vivo. Methods: Impedance spectroscopy, a non-invasive biophysical technique, was used to monitor the effect of iontophoresis on skin and to determine the extent to which this effect was modulated by pretreatment with penetration enhancers of different physicochemical properties. Results: Azone®/propylene glycol and sodium lauryl sulfate had a profound effect on post-iontophoretic skin impedance, considerably amplifying the effect of current passage. Post-iontophoretic impedance spectra of skin pretreated with oleic acid showed a smaller decrease in skin impedance. Neither stearic acid nor linoleic acid had comparable effects, however. For these enhancers, the changes observed were attributable to the vehicle, propylene glycol, which complemented the action of iontophoresis and increased its effectiveness. A parameter, analogous to the membrane time constant, was introduced that enabled the degree of perturbation and the rate of recovery of skin impedance to be gauged. Conclusions: This study provides the first direct in vivo measurements of the interaction between chemical and electrical enhancement, the two principal techniques used to promote transdermal drug delivery. Changes in the reduction of skin impedance induced by the passage of current have been used as indicators of the interaction between an array of diverse penetration enhancers and iontophoresis. The post-iontophoretic impedance data have been fitted to a circuit model involving the parallel arrangement of a resistor and a constant-phase element, and the modulation of the effect of iontophoresis by the penetration enhancers has been determined from changes in the equivalent circuit parameter values.