We report the detection of charge reversal induced by the adsorption of a cationic polyelectrolyte, poly(allylamine) hydrochloride (PAH), to buried supported lipid bilayers (SLBs), used as idealized model biological membranes. We observe changes in the surface potential in isolation from other contributors to the total SHG response by extracting the phase-shifted potential-dependent third-order susceptibility from the overall SHG signal. We demonstrate the utility of this technique in detecting both the sign of the surface potential and the point of charge reversal at buried interfaces without any prior information or complementary techniques<i>.</i>Furthermore, isolation of the second-order susceptibility contribution from the overall SHG response allows us to directly monitor changes in the Stern Layer. Finally, we characterize the Stern and Diffuse Layers over single-component SLBs formed from three different zwitterionic lipids of different gel-to-fluid phase transition temperatures (T<sub>m</sub>s). We determine whether the surface potential changes with the physical phase state (gel, transitioning, or fluid) of the SLB and incorporate 20 percent of negatively charged lipids to the zwitterionic SLB to investigate how the surface potential changes with surface charge.
