Among the optical characterization techniques, the Fourier Transform Infrared spectroscopy (FTIR) can offer high sensibility and accuracy to detect minimal chemical changes into the biological sample and in principle, can be used to differentiate the normal from neoplastic tissues. The aim of the present study is to evaluate the differences on FTIR spectra of the normal cell due to different fixation protocols for histological processing. Immortalized, non-cancerous mice lung epithelial cell line e10 (American Type Culture Collection, Rockville, MD, USA) was maintained in complete e10 culture medium (CMRL-1066 (Gibco), 10% SUF and L-glutamine 200 mM). The cultures were fixed with following substances: 5% formalin in PBS, methacarn (60% methanol, 30% chloroform and 10% acetyl acid), 70% alcohol and also the unfixed cells, were cultured and maintained in PBS. The FTIR spectra were acquired on a Nicolet 6700 (Thermo Scientific Nicolet™, Waltham, MA, USA) spectrophotometer at 4 cm⁻¹ resolution, 30 scans, in the 4000–500 cm⁻¹ spectral range. Nine infrared spectra were obtained from each fixation protocol. Main region of spectra used in the analysis was 1800–800 cm⁻¹. All spectra were analyzed by vector normalization, to determine any possible difference in bands and peak position. These results indicate that all fixing protocol change some of the specific characteristics of FTIR spectra. The formalin fixation was the protocol that caused less modification on the spectra related to the unfixed one. Therefore the choice of the fixing protocol depends on the specific information wanted from the spectra.