Here, we present a protocol for describing the generation of a proximal tubule-on-chip model using human induced pluripotent stem cell (hiPSC)-derived kidney organoids for renal transporter analysis. We describe steps for hiPSC differentiation into kidney organoids, proximal tubule cell isolation, and microfluidic chip seeding. The hiPSC-derived model enhances transporter expression and polarization, with improved uptake and efflux functions compared to conventional immortalized cell-based models. This protocol serves as a platform to assess renal transporter function, nephrotoxicity, and drug-drug interactions. For complete details on the use and execution of this protocol, please refer to Ma et al.(1).