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Fourier-Transform Infrared microspectroscopy (μFT-IR) has been usefully applied in the analysis of the complex biological processes occurring during X-ray radiation-cell interaction. Different experimental approaches are available for FT-IR spectra collection (transmission, attenuated total reflection (ATR), and transflection modes) from cells samples. Recently, some problems have been raised about the role of transmitted and reflected components of the infrared beam in transflection mode. For this reason, we investigated two different transflection approaches for collecting spectra from cells exposed to X-ray. In the former approach, cells were grown on MirrIR slides, and for the second approach, cell pellets were prepared. In both cases, SH-SY5Y neuroblastoma cells were used. X-ray exposure was performed at doses of 2 and 4 Gy. Spectra were obtained by using both the approaches in the 600–4000 cm⁻¹ spectral range from exposed and not-exposed samples. The main contributions from proteins, lipids, carbohydrates, and DNA were clearly evidenced in spectra obtained with the two different acquisition approaches. A comparison among them has been also reported.