Raman spectroscopy is a powerful biochemical analysis technique that allows for the dynamic characterization and imaging of living biological cells in the absence of fluorescent stains. In this review, we summarize some of the most recent developments in the noninvasive biochemical characterization of single cells by spontaneous Raman scattering. Different instrumentation strategies utilizing confocal detection optics, multispot, and line illumination have been developed to improve the speed and sensitivity of the analysis of single cells by Raman spectroscopy. To analyze and visualize the large data sets obtained during such experiments, sophisticated multivariate statistical analysis tools are necessary to reduce the data and extract components of interest. We highlight the most recent applications of single cell analysis by Raman spectroscopy and their biomedical implications that have enabled the noninvasive characterization of specific metabolic states of eukaryotic cells, the identification and characterization of stem cells, and the rapid identification of bacterial cells. We conclude the article with a brief look into the future of this rapidly evolving research area.