Recently, whole-body gamma-knife as a kind of radiotherapy equipment, characterized by precision of targeting irradiation, was applied in clinic. However, there is no conventional treatment regimen suitable for application to whole-body gamma-knife as yet. For providing reference to clinic to determine and optimize the treatment regimens for whole-body gamma-knife, a simple and quick bivariate flow cytometric approach was adopted to evaluate the killing effects of differently fractionated regimens of whole-body gamma-knife radiotherapy on the same cells, in which Annexin V-FITC (fluorescein-isothiocyanate) was used for discriminating apoptotic cells, propidium iodide (PI) for necrotic or dead cells and absolute cell count beads for absolute count of remnant surviving cells to examine the absolute killing effect. The results showed that the different survival rates of the same cells treated with different multifractionated irradiation regimens, relevant to clinical trial, might be sensitively, easily and rapidly evaluated by this approach. The evaluation of absolute killing effect, which is important to clinic, is very difficult to realize by a conventional clonogenic assay. For the approach adopted in the experiments, the cells in the treatment courses were not affected by non-treatment factors, such as cell migration, detachment, reattachment, bystander effect and medium factors encountered in a conventional clonogenic assay, and the conditions were more relevantly similar to cells in vivo. This approach might be used as a rapid cell survival assay especially in evaluating the absolute killing effect of a multifractionated treatment regimen, and also applied in the assessment in cytotoxic killing effect, such as chemotherapy.