Matrigel is widely used for modelling the extracellular (matrix ECM) in invitro epithelialization studies. However, while Matrigel can mimic the native 3D epithelial environment in a physiologically relevant manner, its applications are limited by its tumorigenicity, batch‐batch variability and presence of non‐ defined growth factors. To overcome these shortcomings, we propose a chemically defined soluble BM solution, with the hypothesis that this solution can give comparable results to that of Matrigel in epithelialization studies, and hence can be used as its alternative. Different combinations of Collagen IV, Laminin 511 and Collagen I were combined in variable proportions to form chemically defined soluble BM Compositions. Optimized concentrations of epithelial cells were added on top of the gelated BM layer and cultured for 3‐4 days. The epithelialization of three different cell lines – MDCK, MCF7 and CaCo2, in terms of the barrier integrity of the resultant bi‐layered epithelial samples were then evaluated and compared to that of Matrigel, by means of confocal imaging of barrier markers (e‐cadherin and Zo1) and functional assays such as TEER Measurement and Dextran permeability assay. The preliminary findings of the project demonstrated that the barrier integrity of each of the cell lines were comparable, if not better, to that of Matrigel. The findings of the project can pave the way to optimizing the composition of an ECM solutions that is not only chemically defined, but is also free from tumorigenic components, and hence can be used for epithelialization studies involving both healthy as well as tumor cells.