A bio-impedance chip has been developed for real-time monitoring of the kinetics of epithelial cell monolayers in vitro. The human bronchial epithelial cell line (16-HBE 14o-) was cultured in Transwells creating a sustainable and interactive model of the airway epithelium. Conducting polymer polypyrrole (PPy) doped with polystyrene sulfonate (PSS) was electrochemically deposited onto the surface of gold-plated electrodes to reduce the influence of the electrical double layer on the impedance measurements. Finite element and equivalent circuit models were used to model and determine the electrical properties of the epithelial cell monolayer from the impedance spectra. Electrically tight, confluent monolayers of 16 HBE 14o- cells were treated with increasing concentrations of either Triton X-100 to solubilize cell membranes or ethylene glycol-bis(2-aminoethyl-ether)-N,N,N'N'-tetraacetic acid (EGTA) to disrupt cell-cell adhesion. Experimental impedance data showed that disruption of epithelial barrier function in response to Triton X-100 and EGTA can be successfully measured by the bio-impedance chip. The results were consistent with the conventional hand-held trans-epithelial electrical resistance measurements. Immunofluorescent staining of the ZO-1 tight junction protein in the untreated and treated 16HBEs was performed to verify the disruption of the tight junctions by EGTA.