Dialysis or kidney transplantation is the only therapeutic option for end stage renal disease. Accordingly, there is a large unmet clinical need for new causative therapeutic treatments. Obtaining robust models that mimic the complex nature of the human kidney is a critical step in the development of new therapeutic strategies. Here we establish a synthetic in vitro human renal tubulogenesis model based on a tunable glycosaminoglycan-hydrogel platform. In this system, renal tubulogenesis can be modulated by the adjustment of hydrogel mechanics and degradability, growth factor signaling, and the presence of insoluble adhesion cues, potentially providing new insights for regenerative therapy. Different hydrogel properties were systematically investigated for their ability to regulate renal tubulogenesis. Hydrogels based on heparin and matrix metalloproteinase cleavable peptide linker units were found to induce the morphogenesis of single human proximal tubule epithelial cells into physiologically sized tubule structures. The generated tubules display polarization markers, extracellular matrix components, and organic anion transport functions of the in vivo renal proximal tubule and respond to nephrotoxins comparable to the human clinical response. The established hydrogel-based human renal tubulogenesis model is thus considered highly valuable for renal regenerative medicine and personalized nephrotoxicity studies. Statement of Significance The only cure for end stage kidney disease is kidney transplantation. Hence, there is a huge need for reliable human kidney models to study renal regeneration and establish alternative treatments. Here we show the development and application of an in vitro human renal tubulogenesis model using heparin-based hydrogels. To the best of our knowledge, this is the first system where human renal tubulogenesis can be monitored from single cells to physiologically sized tubule structures in a tunable hydrogel system. To validate the efficacy of our model as a drug toxicity platform, a chemotherapy drug was incubated with the model, resulting in a drug response similar to human clinical pathology. The established model could have wide applications in the field of nephrotoxicity and renal regenerative medicine and offer a reliable alternative to animal models.