In this work we investigate the capability of Raman microscopy (RM) to detect inherent sources of biochemically based spectral variability between single cells of a human tumor cell line (DU145) cultured in vitro. Principal component analysis (PCA) is used to identify differences in single-cell Raman spectra. These spectral differences correlate with (1) cell cycle progression and (2) changing confluency of a cell culture during the first 3 to 4 days after sub-culturing. Cell cycle regulatory drugs are used to synchronize the cell cycle progression of cell cultures, and flow cytometry is used to determine the cell cycle distribution of cell cultures at the time of Raman analysis. Spectral variability arising from cell cycle progression is (1) expressed as varying intensities of protein and nucleic acid features relative to lipid features, (2) well correlated with known biochemical changes in cells as they progress through the cell cycle, and (3) shown to be the most significant source of inherent spectral variability between cells. Furthermore, the specific biomolecules responsible for the observed spectral variability due to both cell cycle progression and changes in cell culture confluency can be identified in the first and second components of principal component analysis (PCA). Our characterization of the inherent sources of variability in Raman spectra of single human cells will be useful for understanding subtle spectral differences in RM studies of single cells.