E-cigarettes have been suggested as a potentially healthier alternative to cigarettes based on studies using cell viability, DNA damage, and transcriptional response assays. However, little is known about the effect of e-cigarette aerosols on the integrity of the tracheal epithelium, specifically with respect to barrier resistance. This is partly due to the lack of methods for monitoring epithelia at the air-liquid interface (ALI), i.e., under physiological conditions. Here, it is shown that an organic electrochemical transistor can be adapted for the measurement of barrier resistance at the ALI. This technology enables accurate, continuous quantification of tracheal barrier integrity through the use of a conformable gate electrode placed on top of the cell-secreted mucus, obviating the need for addition of culture medium or buffer as a conductance medium for rigid electrodes. This platform allows for the detection of a dose-dependent, rapid decrease in barrier resistance of an in vitro model of human bronchial epithelium (MucilAir) after E-cigarette aerosols exposure. The system represents a powerful tool to study tissue responses of the human airway epithelium to inhaled smoke. The same technology will have broad applications for toxicology studies on other tissues with ALI, including other airway tissues and skin.