To establish and validate a method for cell irradiation in 96-well and 6-well plates using a linear accelerator,three irradiation methods (G0B0F40,G0B1.5F40,and G180B1.5F40) were designed to irradiate cell culture plasticware simulated with RW3 slab phantom and polystyrene.The difference between the actual physical measured dose and the preset dose was compared among the three methods under the preparatory conditions of 2,4,6,8,and 10 Gy.MDA-MB-231 cells were analyzed by using a cell proliferation assay and a clonogenic assay to verify the difference between the three cell irradiation methods on cell radiosensitivity.For each preset dose,the difference between the actual measured dose and the preset dose was the lowest for Method G0B1.5F40,the second lowest for Method G180B1.5F40,and the maximum for Method G0B0F40.The ranges of the differences were -0.28 to 0.02％,-2.17 to-1.80％,and-4.92 to -4.55％,and 0.31 to-0.12％,-3.42 to-2.86％,and -7.31 to-6.92％,respectively,for 96-well and 6-well plates.The cell culture experiments proved that Method G0B1.5F40 was an accurate,effective,simple,and practical irradiation method.The most accurate and effective cell irradiation method should always be used,as it will reduce dose differences and instability factors and provide improved accuracy and comparability for laboratories researching cellular radiosensitivity.