- In last decades infrared spectroscopy has demonstrated potential as a novel technology for early cancer diagnosis. Among the various IR spectroscopic techniques special interest has arisen from methods based on evanescent wave absorbance due to the possibility for in situ and in vivo implementation. The goal of the present study is to examine the potential of Attenuated Total Reflectance (ATR) spectroscopy for early detection of premalignant changes. As a model we used both cell lines and primary cells, which were transformed to be malignant by a retrovirus. Spectral measurements were performed at various post infection stages in parallel with morphological observations. Our results showed gradual and consistent spectral alterations in both cell cultures due to carcinogenesis, which were outlined using Principal Component Analysis (PCA). The main spectral differences appeared in three spectral ranges: at 3000–2800 cm−1 (attributed to stretching vibrational modes of lipids and proteins), at 1470–1300 cm−1(attributed to bending overlapping modes of lipids and proteins) and also at the highly overlapping spectral range at 1000–1200 cm−1 (attributed to bending and starching vibrational modes corresponding to all types of biological macromolecules). In order to obtain robust unsupervised classifications of the malignant progression we applied approaches of Linear Discriminant Analysis (LDA). The classifications based on Mahalanobis distances allowed us to discern that the accuracy of successful identification of premalignant stages varied between 86.5–97.2%. Our results show that ATR spectroscopy in tandem with proper statistical tools may provide a promising technique for early detectable signals of malignant progression.