Determination of neoplasia is largely dependent on the state of cell growth. Infrared (IR) spectroscopy has the potential to measure differences between normal and cancerous cells. When analyzing biopsy sections using IR spectroscopy, careful analyses become important since biochemical variations may be misinterpreted due to variations in cell cycle. Processes like DNA replication, transcription and translation to produce proteins are important in determining if the cells are actively dividing but no studies on this aspect using IR spectroscopy have been conducted on isolated cell nuclei. Nuclei hold critical information about the phase of cell and its capacity to divide, but IR spectra of nuclei are often confounded by cytoplasmic signals during data acquisition from intact cells and tissues. Therefore, we sought to separate nuclear signals from cytoplasmic signals and identify spectral differences that characterize different phases of the cell cycle. Both cells and isolated nuclei were analyzed to assess the effect of the cytoplasmic background and to identify spectral changes in nuclei in different phases of cell cycle. We observed that signals of DNA could be obtained when imaging nuclei isolated from cells in different phases of cell cycle, which is in contrast to the oft-cited case in cells wherein nuclear contributions are obscured. The differences across cell cycle phases were more pronounced in nucleic acid regions of the spectra, showing that the use of nuclear spectrum can provide additional information on cellular state. These results can aid in developing computational models that extract nuclear spectra from whole cells and tissues for more accurate assessment of biochemical variations.