We describe the first microfluidic device for in vitro testing of brachytherapy (BT), with applications in translational cancer research. Our PDMS-made BT-on-chip system allows highly precise manual insertion of clinical BT seeds, reliable dose calculation using standard clinically-used TG-43 formalism and easy culture of naturally hypoxic spheroids in less than 3 days, thereby increasing the translational potential of the device. As the BT-on-chip platform is designed to be versatile, we showcase three different gold-standard post-irradiation bioassays and recapitulate, for the first time on-chip, key clinical observations such as dose rate effect and hypoxia-induced radioresistance. Our results suggest that BT-on-chip can be used to safely and efficiently integrate BT and radiotherapy to translational research and drug development pipelines, without expensive equipment or complex workflows.