In this work, we report the development of a measurement chamber linked with a quadrupole mass spectrometer (QMS) for in situ investigation of the effect of thin film cracking on the gas permeation of coated flexible polymeric substrates. The chamber enables the establishment of a bulged state of the substrate/coating system, which causes the cracking of the coating layer. The increase in the gas permeation rate due to the presence of cracks can be monitored precisely using the QMS without movement or re-clamping of the samples between each measurement step. This method eliminates the probability of uncontrollable mechanical changes in the sample, and with the mass spectrometer, high sensitivity, reliability, and reproducibility of the experimental data become available.