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The ability of Fourier transform infrared (FTIR) spectroscopy in analyzing cells at a molecular level was exploited for investigating the biochemical changes induced in protein, nucleic acid, lipid, and carbohydrate content of cells after irradiation by graded X-ray doses. Infrared spectra from in vitro SH-SY5Y neuroblastoma cells following exposure to X-rays (0, 2, 4, 6, 8, 10 Gy) were analyzed using a ratiometric approach by evaluating the ratios between the absorbance of significant peaks. The spectroscopic investigation was performed on cells fixed immediately (t₀ cells) and 24 h (t₂₄ cells) after irradiation to study both the initial radiation-induced damage and the effect of the ensuing cellular repair processes. The analysis of infrared spectra allowed us to detect changes in proteins, lipids, and nucleic acids attributable to X-ray exposure. The ratiometric analysis was able to quantify changes for the protein, lipid, and DNA components and to suggest the occurrence of apoptosis processes. The ratiometric study of Amide I band indicated also that the secondary structure of proteins was significantly modified. The comparison between the results from t₀ and t₂₄ cells indicated the occurrence of cellular recovery processes. The adopted approach can provide a very direct way to monitor changes for specific cellular components and can represent a valuable tool for developing innovative strategies to monitor cancer radiotherapy outcome.