Fourier transform infrared microspectroscopy (FTIR-MSP) is potentially a powerful analytical method for identifying the spectral properties of biological activity in cells. The goal of the present research is the implementation of FTIR-MSP to study early spectral changes accompanying malignant transformation of cells. As a model system, cells in culture are infected by the murine sarcoma virus (MuSV), which induces malignant transformation. The spectral measurements are taken at various postinfection time intervals. To follow up systematically the progress of the spectral changes at early stages of cell transformation, it is essential first to determine and validate consistent and significant spectral parameters (biomarkers), which can evidently discriminate between normal and cancerous cells. Early stages of cell transformation are classified by an array of spectral biomarkers utilizing cluster analysis and discriminant classification function techniques. The classifications indicate that the first spectral changes are detectable much earlier than the first morphological signs of cell transformation. Our results point out that the first spectral signs of malignant transformation are observed on the first and third day of postinfection (PI) (for NIH/3T3 and MEF cell cultures, respectively), while the first visible morphological alterations are observed only on the third and seventh day, respectively. These results strongly support the potential of developing FTIR microspectroscopy as a simple, reagent-free method for early detection of malignancy.