(1) Breast cancer is presently the leading cause of death in women worldwide. This study aims at identifying molecular biomarkers of cancer in human breast cancer cells, in order to differentiate highly aggressive triple-negative from non-triple-negative cancers, as well as distinct triple-negative subtypes, which is currently an unmet clinical need paramount for an improved patient care. (2) Raman and FTIR (Fourier transform infrared) microspectroscopy state-of-the-art techniques were applied, as highly sensitive, specific and non-invasive methods for probing heterogeneous biological samples such as human cells. (3) Particular biochemical features of malignancy were unveiled based on the cells’ vibrational signature, upon principal component analysis of the data. This enabled discrimination between TNBC (triple-negative breast cancer) and non-TNBC, TNBC MSL (mesenchymal stem cell-like) and TNBC BL1 (basal-like 1) and TNBC BL1 highly metastatic and low-metastatic cell lines. This specific differentiation between distinct TNBC subtypes—mesenchymal from basal-like, and basal-like 1 with high-metastatic potential from basal-like 1 with low-metastatic potential—is a pioneer result, of potential high impact in cancer diagnosis and treatment.