Background: Recently, the Flattening Filter Free (FFF) beams, which allow a dose rate increase of up to four times compared to the normal dose rate, have been incorporated into radiation therapy machines. The aim of this study is to evaluate antitumor activity in hypoxic cells irradiated with different dose rates using FFF beams and to identify the casual mechanism for cellular damage during irradiation.  Materials and Methods: EMT6 cells were treated with 95% N₂ and 5% CO₂ to maintain a hypoxic condition. Three dose rates, namely, 6.27 Gy/min, 12.00 Gy/min, and 18.82 Gy/min, were used to deliver the prescribed dose of 2 to 4 Gy using the TrueBeam linear accelerator. The number of colonies was counted to evaluate the cell surviving fraction. To investigate the mode of action, additional experiments to detect reactive oxygen species (ROS) by aminophenyl fluorescein (APF) assay, and DNA double-strand breaks (DSBs) by γH2AX assay were performed. Results: Irradiation of hypoxic cells using FFF beams increases antitumor activity as a function of dose rate. The fluorescence of the APF assay was significantly increased when high dose rates were used. In addition, results from our γH2AX assays show that the number of DNA DSBs increased as a function of dose rate, in hypoxic cells. Conclusion: We demonstrate that there is a significant dose rate-dependent difference in antitumor activity in hypoxic cells, when FFF beams are used.