The glomerular basement membrane has a complex composition and structure in vivo. In vitro models of the glomerular capillary wall have been developed using microfluidic and perfusion systems, however, how the extracellular matrix alters under static and flow conditions has not yet been investigated. Here we performed proteomic analyses following a previously described workflow (Byron et al., 2014; Rashid et al., 2012; Soteriou et al., 2013) to investigate the protein composition of podocyte and glomerular endothelial coculture cell-derived matrix and whether this was altered when cells experienced shear stress induced by flow. Our results reveal a distinct separation between flow and static-treated samples and highlight compositional changes in the matrix in the microfluidic in vitro model, and confirm the flow impacted the abundance of certain matrix proteins.