p53 is a crucial tumor suppressor and plays an important role in cell cycle arrest, DNA damage repair, promotion of cell senescence and apoptosis, prevention of DNA damage and maintaining genomic stability and integrity. It has been reported that p53 might also be related to radiation sensitivity, for which the involved effects and processes could be further examined biochemically at the molecular level. In this study, we explored a new spectroscopic approach to probe the radiation-induced biological effects related to p53. Infrared microspectroscopy was used to detect the metabolic changes related to p53 under particle radiation. After alpha-particle irradiation of HCT116 cells (p53 ^/ , p53^–/–), cell cycle arrest, DNA damage and lipid peroxidation in the cancer cells were observed using Fourier-transform infrared (FTIR) spectroscopy and microspectroscopy imaging. A remarkable difference in radiosensitivity between the two genotypes of cells was observed as well. This work provides a biochemical analysis of the p53-related radiation effects in cells and demonstrates the potential usefulness of FTIR microspectroscopy in the field of radiation research.