The exposure of animals or cultured cells to radiation is the essential and common step in experimental researches to elucidate biological effects of radiation. When an X-ray generator is used as a radiation source, physical parameters including dose, dose rate, and the energy spectrum of X-ray play crucial roles in biological outcome. Therefore, those parameters are the important points to be checked in quality control and to be carefully considered in advance to the irradiation to obtain the accurate and reproductive results. Here we measured radiation dose emitted from the X-ray irradiator for research purposes by using clonogenic survival of cultured mammalian cells as a biological marker in parallel with physical dosimetry. The results drawn from both methods exhibited good consistency in the dose distribution on the irradiation stage. Furthermore, the close relationship was observed between cell survival and the photon energy spectrum by using different filter components. These results suggest that biological dosimetry is applicable to quality control of X-ray irradiator in adjunct to physical dosimetry and that it possibly helps better understanding of the optimal irradiating condition by X-ray users in life-science field.