Raman spectroscopy has proven to be a very powerful technique and is currently experiencing a renaissance. In this paper, it is used to explore the interaction between doxorubicin and malignant hepatocytes in vitro. For the addition of doxorubicin, the band intensity at 1609 cm-1, mainly assigned to C=C in-plane bending mode of phenylalanine and/or tyrosine residues, increases significantly, and the intensities of the bands at 1585 and 1313 cm-1, mainly due to the guanine bases, decrease greatly. In addition, Raman spectra are investigated at different doxorubicin concentrations, and the mean areas ratios of the band at 1450 to that at 1003 cm-1, A1450/A1003, fluctuate according to the doxorubicin concentration increasing, which suggests that doxorubicin affects the relative content of lipid in cells.