Polymethoxyflavones (PMFs), especially Nobiletin (NBT) and 5-demethylnobiletin (5DN), have been widely studied and proved to have anti-cancer effects. However, their exact modes of action against cancer cells are not fully understood. Raman spectroscopy is a molecular vibrational spectroscopy can be used to measure overall biochemical function of single cells noninvasively and without adding any chemical labels. Herein, we characterized the cellular responses of human colorectal cancer HT29 and HCT116 cells, that grow on the gold coated slides, to the treatments of NBT and 5DN using Raman microscopy. Raman microscopic images obtained indicated the different modes of action of these two PMFs against colorectal cancer cells. NBT induced more changes in the nucleic acid peaks and protein peaks, while 5DN induced more changes in the localized lipid peaks. We further analyzed both attached and floating cells in response to NBT and 5DN treatments using Raman microscopy and principal component analysis. Result showed large spectral heterogeneity of floating cells compared to attached cells, particular in the range for protein spectral profiles. In response to the treatment s, the difference between the floating cells and attached cells became smaller over time. This indicates the different sensitivity and behavior of individual cells to the treatment. Identification of individual cancer stem cells (CSC) from normal cancer cells is challenging. We applied Raman microscopy to identify and characterize CSC. CSC were enriched in non-attachable petri-dish to form cell spheres. The spheres containing both CSC and normal cancer cells and could be separated based on the density. It is interesting that 70% of CSC enriched density layer produced a distinct Raman peak at around 1049 cm-1, while almost no normal cells contained this peak. This finding demonstrates the 1049 cm-1 peak might be used as indicator peak for CSC. Further validation is needed. Overall, our research demonstrates the feasibility of Raman microscopy to characterize individual cells and to investigate the heterogeneous response of the cell population to treatment. Moreover, there is a great potential of using Raman microscopy for identify individual CSC from normal cancer cells and investigate their heterogeneous response to the treatment.