Epithelial and endothelial barrier function is typically studied in vitro by growing the cells of interest on permeable supports that are sandwiched between two fluid compartments. This setup mimics the physiological situation with the cell layer as the diffusion barrier at the interface between two chemically distinct fluids. Routinely, the barrier function is quantitatively described by two key parameters: (i) the transepithelial or transendothelial electrical resistance (TER) as a measure of the permeability for small inorganic ions and (ii) the permeability coefficient (PE) as a descriptor of the permeability for molecular tracers. So far the two parameters have been determined in separate experiments. This study introduces a device that allows for simultaneous detection of PE and TER of the very same cell monolayer in one single experiment (PETER-assay). The novel approach is entirely based on AC impedance measurements in two different modes, so that TER and PE become available in real time. The new approach is demonstrated for three epithelial cell lines derived from the kidney (MDCK-I, MDCK-II, NRK) with very different barrier properties under stationary conditions and when challenged by barrier-breaking fungal toxin cytochalasin D. PETER provides an excellent time-resolution and completely automated data collection.