Epithelial dysfunction and subepithelial fibrosis in the airways are pathophysiological features that indirectly promote airflow obstruction in asthma. Respectively, these changes result in impaired epithelial barrier function and airway stiffening. I hypothesize that subepithelial matrix stiffening disrupts airway epithelial barrier function. I engineered an experimental platform to replicate airway stiffening using lung epithelial cell lines. This platform allowed measurement of barrier function (permeability assay), in addition to traditional measurements of mRNA abundance (qPCR), protein abundance (Western blot) and protein localization (immunofluorescence microscopy). Substrate stiffness did not impact barrier-related gene and protein markers but did regulate their subcellular locations. Moreover, I observed differential responses of barrier function in response to transforming growth factor β across different substrate stiffnesses. These proof-of-concept results demonstrate substrate stiffness may regulate epithelial barrier function in a disease-relevant manner, and that my platform can be used to explore how airway stiffening regulates airway epithelial function in asthma.