Cancer stem cells arising from accumulated mutations in stem cells and progenitor cells are highly responsible for recurrence, resistance to therapy, and metastasis of cancer though they are a small percentage of cancer cells. Malignant melanoma is a common type of cancer and its incidence rate, mortality, and morbidity have increase alarmingly. The goal of this study is to investigate cancer stem cells in malignant melanoma model using vibrational spectroscopy. Vibrational spectroscopy confers both qualitative and quantitative information regarding the molecules which means both biochemical composition of cells and concentrations of biomolecules can be determined with vibrational spectroscopy. Moreover, relationships between the experimental groups and statistical importance of spectral data can be evaluated by applying multivariate analysis methods to collected spectra. Vibrational spectroscopy i.e. infrared and Raman spectroscopy has been widely used in many cellular and other biological studies, obtaining successful results. In this work, cancer stem cells, non-stem cancer cells, and bulk population of cancer cells were analysed with infrared and Raman spectroscopy and recorded data were analysed with multivariate methods at 11th, 24th, 48th, and 72nd hours of cell cycle. As a result, biochemical and biophysical differences between three groups at different hours were determined, and information related to cell cycle of cells were obtained. This study supports that vibrational spectroscopy can be used in the characterization of cells and combined with multivariate analyses information gathered from spectra can be enhanced significantly.