Assessing the tumor invasiveness is a paramount diagnostic step to improve the patients care. Infrared spectroscopy access the chemical composition of samples; and in combination with statistical multivariate processing, presents the capacity to highlight subtle molecular alterations associated with malignancy development. Our investigation demonstrated that infrared signatures of cell lines presenting various invasiveness phenotypes contain discriminant spectral features, which are useful informative signals to implement an objective invasiveness scale. This last development reflects the interest of vibrational approach as a candidate biophotonic label-free technique, usable in routine clinics, to characterize quantitatively tumor aggressiveness. In addition, the methodology can reveal the heterogeneity of cancer cells, opening the way to further researches in cancer science.