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This method describes a novel approach to systematically investigate the effect of the fluid shear stress (FSS) on epithelial cells thanks to a single microfluidic device based on Hele-Shaw geometry. The method was validated with intestinal Caco-2 cell monolayers and lung A549 cells. We provide guidelines to adjust the experimental parameters to apply specific ranges of FSS and to specify more accurately the area where to image the cells within the device by the performance of a computational simulation of the fluid flow. Most importantly, this simulation enables to validate the equation. This approach was successfully applied to systematically investigate Caco-2 cell monolayers-based intestine-on-chip models as reported in a companion article published in Biomaterials. This study showed that exposure to microfluidic FSS induces significant phenotypical and functional changes. A detailed understanding of the effects of the FSS will enable the realization of in vitro organs-on-chip models with well-defined characteristics tailored to a specific purpose. The Hele-Shaw approach used in this study could be readily applied to other cell types and adapted for a wide range of physiologically relevant FSS.•Fluid shear stress is a key parameter in the differentiation of epithelial cells cultured in organ-on-chip models.•A simple approach can be used to assess the effect of fluid shear on cellular monolayer cultured in microfluidic devices.•Careful optimization of fluid shear stress environment is necessary for the development of better-defined organ-on-chip models.•Computational simulation of the fluid flow gives an accurate definition of the FSS in a microfluidic channel necessary to interpret the results., Graphical abstract Image, graphical abstract