Despite an enormous investment of clinical and financial resources, chronic kidney disease (CKD) remains a global health threat. The lack of reliable in vitro systems that can efficiently mimic the renal and glomerular environment has hampered our ability to successfully develop novel and more renal specific drugs. Even though some success in generating in vitro tubule analogues and kidney organoids has been described, a major challenge remains for the in vitro assembly of the filtration unit of the kidney, the glomerulus. We have recently developed a novel glomerulus-on-a-chip system that mimics the characteristic and functionality of the glomerular filtration barrier, including its response to injury. This system recapitulates the functions and structure of the in vivo glomerulus, including permselectivity; indeed, we have confirmed free diffusion of insulin as well as impermeability to physiological concentrations of albumin. Exposure to nephrotoxic agents like puromycin aminonucleoside leads to a significant increase in albumin leakage. When exposed to sera from patients with anti-podocyte autoantibodies, the chip shows albumin leakage to an extent proportional to in vivo clinical data, phenomenon not observed with sera from either healthy controls, confirming functional response to injury. We describe here the detailed procedure to obtain a glomerulus-on-a-chip system that replicates both phenotypically and functionally the in vivo glomerular microenvironment.