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We report here the use of Fourier transform infrared spectroscopy (FTIR), ultraviolet-visible (UV-vis) absorption spectroscopy, and flow cytometry (FCM) to analysis the best dose of X-ray for human laryngeal squamous cell carcinoma cell lines (Hep-2). Our analysis indicates specific FTIR and UV-vis spectral differences between X-irradiated and normal Hep-2 cells. In addition, striking spectral differences are seen in FTIR spectra in the ratios at 2925/2958 and 1654/1542 cm(-1). These two ratios of the X-irradiated cells for 8 Gy dose group with value of 1.07+/-0.025 and 1.184+/-0.013, respectively, were more notable (mean+/-S.D., n=5, P<0.05) compared with that of the cells for the controls. UV-vis absorption spectra analysis shows X-ray irradiation disturbed the metabolism of phenylalanine and tyrosine intracellular, maybe, which was caused by cell cycle arrest. Spectroscopy analysis suggests 8 Gy is a better dose of X-ray for lowering the canceration degree of Hep-2 cells. Moreover, FCM analysis shows the apoptosis of X-irradiated cells depended on the radiation dose to some extent, but it was not linear. The total apoptosis ratio with value of (20.793+/-1.133)% (P<0.01, n=5) for the 12 Gy dose group was the maximum, however, the maximum apoptosis ratio per Gray (total apoptosis ratio/radiation dose) was the cells of the 2 Gy dose group with value of (4.887+/-0.211)% (P<0.05, n=5). Our data suggest that Hep-2 cells are given 2 Gy radiation of X-ray once a time, 8 Gy per week (accumulatively), the effect for lowering the canceration degree and restraining the proliferation of Hep-2 cells will be better.