The renal proximal tubule is a primary site of excretion and reabsorption of endogenous and exogenous small molecules and a critical determinant in pharmacokinetic modeling. Because of the dynamic nature of the transport processes in the renal proximal tubular epithelial cells (RPTEC), that depend on sheer stress and correct polarization, in vitro modeling of this tissue is challenging. A number of microphysiological systems for studies of kidney proximal tubule have been proposed, and one such model is the PhysioMimix™ T12 (CNBio) where RPTEC can be cultured on the bottom of Transwells and exposed to media at a constant flow rate of up to 2.5 µL/s. This model has a wide application potential for testing of both pharmaceuticals and other chemicals. Therefore, as part of TEX-VAL Consortium, a multi-stakeholder effort for stablishing the functionality, reproducibility, robustness, and reliability of microphysiological systems, this study aimed to evaluate the PhysioMimix™ T12 platform using different RPTEC cell types and conditions. RPTEC/TERT1 cells (parental and OAT1, OCT2, and OAT3 overexpressing lines) were cultured in Transwells® and grown under typical static conditions or placed into the CNBio PhysioMimix™ T12 plate, where they were exposed to flow to allow for direct shear stress. Experiments were performed for 7 days under static or dynamic conditions and basal function was compared: including transepithelial resistance, water transport, and transporter expression/localization. Additionally, bi-directional transport of cisplatin, tenofovir, paraaminohippuric acid, and perfluorooctanoic acid was tested in the presence or absence of probenecid (OAT1-inhibitor) in parental and OAT1 cells under static and dynamic conditions. We demonstrate that barrier function as well as water and chemical transport were more physiological under flow conditions. Additionally, the presence of fluid shear stress significantly increased AQP1 protein expression, concordant with an increase in water transport. These results demonstrate that a functional proximal tubular model can be established in the PhysioMimix™ T12 platform and be used to study human clearance of both pharmaceuticals and environmental compounds.