Background Ionotropic glutamate receptors AMPAR and NMDAR modulate proliferation, invasion and radioresistance in glioblastoma (GB). Pharmacological targeting is difficult as many in vitro-effective agents are not suitable for in patient applications. We aimed to develop a method to test the well tolerated AMPAR- and NMDAR-antagonist xenon gas as radiosensitizer in GB. Methods We designed a diffusion-based system to perform the colony formation assay (CFA), the radiobiological gold standard, under xenon exposure. Stable and reproducible gas atmosphere was validated with oxygen and carbon dioxide as tracer gases. After checking for AMPAR and NMDAR expression via immunofluorescence staining we performed the CFA with the glioblastoma cell lines U87 and U251 as well as the non-glioblastoma derived cell line HeLa. Xenon was applied after irradiation and additionally tested in combination with NMDAR antagonist memantine. Results The gas exposure system proved compatible with the CFA and resulted in a stable atmosphere of 50% xenon. Glutamate receptor subunits were present in glioblastoma-derived and to a lesser extent in HeLa cells. Significant radiosensitization by xenon was shown in U251 and U87 at irradiation doses of 4–8 Gy and 2–8 Gy, respectively (p < .05). The effect was further enhanced by the addition of memantine, showing significant reduction in the surviving fraction already at 2 Gy for both glioblastoma cell lines (p < .05). Xenon did not increase the radiosensitivity of HeLa cells until a radiation dose of 8 Gy. Conclusion The developed system allows for testing of gaseous agents with CFA. As a proof of concept, we have, for the first time, unveiled radiosensitizing properties of xenon gas in glioblastoma.