- Background Cancer is one of the leading worldwide causes of death. It may be induced by a variety of factors, including carcinogens, radiation, genetic factors, or DNA and RNA viruses. The early detection of cancer is critical for its successful therapy, which can result in complete recovery from some types of cancer. Methods Raman spectroscopy has been widely used in medicine and biology. It is a noninvasive, nondestructive, and water-insensitive technique that can detect changes in cells and tissues that are caused by different disorders, such as cancer. In this study, Raman spectroscopy was used for the identification and characterization of murine fibroblast cell lines (NIH/3T3) and malignant fibroblast cells transformed by murine sarcoma virus (NIH-MuSV) cells. Results Using principal component analysis and LDA it was possible to differentiate between the NIH/3T3 and NIH-MuSV cells with an 80–85% success rate based on their Raman shift spectra. Conclusions The best results for differentiation were achieved from spectra that were obtained from the rich membrane sites. General significance Because of its homogeneity and complete control of most factors affecting its growth, cell culture is a preferred model for the detection and identification of specific biomarkers related to cancer transformation or other cellular modifications.