Radiation therapy is widely used as a treatment tool for malignancies. However, radiation-related complications are still unavoidable risks for off-target cells. Little is known about radiation therapy's possible effects on mechanical features of the off-target cells such as human red blood cells (RBCs). RBCs are nucleus-free circulating cells that can deform without losing functionality in healthy conditions. Thus, to evaluate in vitro effects of radiation therapy on the healthy plasma membrane of cells, RBCs were selected as a primary test model. RBCs were exposed to clinically prescribed radiotherapy doses of 2 Gy, 12 Gy and, 25 Gy, and each radiotherapy dose group was compared to a non-irradiated group. Cells were characterized by stretching using dual-beam optical tweezers and compared using the resulting deformability index. The group receiving the highest radiation dose was found statistically distinguishable from the control group (DI_0Gy = 0.33 ± 0.08), and revealed the highest deformability index (DI_25Gy = 0.38 ± 0.11, p = 0.0068), while no significant differences were found for 2 Gy (DI_2Gy = 0.33 ± 0.08, p = 0.9) and 12 Gy (DI_12Gy = 0.31 ± 0.09, p = 0.2) dose groups. Based on these findings, we conclude that radiotherapy exposure may alter the deformability of red blood cells depending on the dose amount, and measurement of deformability index by dual-beam optical tweezers can serve as a sensitive biomarker to probe responses of cells to the radiotherapy.