Raman spectroscopy has been widely used in biology and medicine for the analysis of biochemical components such as nucleic acids, proteins, and lipids. We exploited the application of the Raman spectroscopy to the monitoring of the process of cell differentiation. Human promyelocytic leukemia (HL‐60) cells were differentiated into granulocytes by dimethyl sulfoxide (DMSO) treatment. Raman spectra, in parallel with the changes of morphology and antigen expression of cells, were analyzed in the cells during differentiation. We found that there is a correlation between the differential stages and Raman characteristic regions related to macromolecular substances. Furthermore, we employed the support vector machine to distinguish the spectra of undifferentiated and differentiated HL‐60 cells and found that the average classification accuracy exceeded 84%. The results suggested that Raman spectroscopy may have potential application in monitoring cell differentiation.