Abstract <p>Cells integrate multiple mechanical cues simultaneously, yet most <i>in vitro</i> models examine extracellular matrix (ECM) stiffness and fluid shear stress (FSS) in isolation, limiting our understanding of mechanotransduction. We developed a parallel plate flow chamber with a polyacrylamide (PAA) substratum enabling independent, tunable control of substrate stiffness and FSS using readily available materials. We confirm that the PAA substratum has controllable mechanical properties that support the growth of Madin-Darby canine kidney epithelial cells across a range of stiffnesses. Furthermore, the flow chamber design accommodates the volumetric equilibrium swelling of the gel, maintaining a predictable fluid channel height that allows for the application of controlled fluid shear stress to cells within the device, confirmed through particle image velocimetry of perfused microspheres. Single flow chambers support the growth of sufficient cellular numbers for endpoint analyses, such as Western blots. Finally, quantitative analysis of F-actin organization revealed that substrate stiffness and FSS synergistically increase filament length with independent effects on filament width, demonstrating the ability and usefulness of this model as a tool for studying the effect of multiple concurrent forces on cell behavior.</p> Graphical abstract <p></p>