Single-cell micro-Raman spectroscopy has been applied to explore cell differentiation in single, live, and malignant cells from two tumor cell lines. The spectra of differentiated cells exhibit substantial enhancement primarily in the intensities of protein peaks with concomitant decrease in intensities of O−P−O asymmetric stretching peaks in DNA/RNA. Principal component analyses show that the spectral score of differentiated cells tends to asymptotically approach that of spectra obtained from normal neural stem cells/progenitors. This lends credence to the notion that the observed spectral changes are specific to differentiation, since upon differentiation, malignant cells become less malignant and tend toward benignity.