The aim of this study was to establish a method to directly assess in vivo the epidermal barrier function by elec. impedance (EI) spectroscopy. Mice epidermal barrier was damaged by epicutaneous application of proteases and cholera toxin and by tape stripping. EI and transepidermal water loss (TEWL) were measured before and after the application. Addnl., histol. anal., immunofluorescence staining, and RT-PCR were performed on skin biopsies to evaluate the epithelial barrier. A few hours after papain application, a dose-dependent reduction of EI was detected, reflecting the decreased barrier function. At the same time, an increase of TEWL was observed, with a significant neg. correlation with EI, demonstrating that EI changes were directly linked to barrier defects. Twenty-four and 48 h after the treatment, EI starts to increase to background levels, indicating tissue healing and restoration of skin barrier. Barrier disruption was confirmed by histol. anal. showing an impaired stratum corneum and higher cellular infiltration after papain application. In addition, immunofluorescence staining and RT-PCR showed downregulation of mols. involved in the barrier function, such as filaggrin, occludin, and claudin-1, and mRNA levels of filaggrin, loricrin, and involucrin. Comparable results were observed after tape stripping and cholera toxin treatment. Elec. impedance spectroscopy is a rapid and reliable diagnostic tool to detect skin barrier defects.