There are a number of powerful techniques for the characterization of cells, for example, immunofluorescence/immunochemistry, which can be used to characterize specific cell surface markers of embryonic stem (ES) cells. However, these techniques have limitations in studying individual living cells, their communication, growth and development. Here we utilize Raman microspectroscopy imaging as a non-invasive tool for rapid analysis of individual cells without the use of external labels to characterize ES cell colonies and to monitor ES cells differentiation during maturation. Stem cell research offers great potential for a wide variety of therapies such as cancer treatments, tissue engineering, as well as many others. Understanding ES cell development and proliferation could provide information about the regulation of embryonic development. This can also render useful information on genotype manipulation of ES cells which would be valuable in disease diagnosis and therapy. Raman microspectroscopy which probes the sub-cellular biochemical environment allows us to monitor changes of cellular components including proteins, lipids, nucleic acids, and carbohydrates. Moreover this technique in synergy with multivariate data analysis methods such as Principal Component analysis (PCA), Hierarchical cluster analysis (HCA) and Vertex Component analysis (VCA) has been shown to be a powerful approach for analyzing hyperspectral data sets. C57BL/6 ES cell line purchased from Open Biosystems was used in the reported study. ES cell colonies maintained undifferentiated and then induced to differentiate according to ATCC standard protocol were fixed with formaldehyde, washed with PBS and analyzed. All spectra and images were collected using a WITec, Inc. Confocal Raman Microscope with the 488 nm laser radiation that offers advantages over excitation in the red/ near-infrared regions in the spectrum (633/785 nm); among them a 6-foled signal enhancement and higher detector sensitivity due to the shorter wavelength. The data obtained provides qualitative information with better spatial resolution comparable to visible light microscopy. We show that this technique is capable of imaging cellular organelles such as the nucleus, variations within the cytoplasm, lipid bodies and extracellular matrix within undifferentiated and differentiated cell colonies. All three multivariate methods easily distinguish the cellular environment demonstrating the potential of detecting diverse sub-cellular features. Furthermore, we attempted to image ES cells differentiated via embryoid body (EB) formation over 9 day time period that gives us a clear picture of biochemically distinct environments within the plated EBs. The results demonstrate that confocal Raman microspectroscopy coupled to aforementioned multivariate methods of analysis can distinguish the intracellular components by their biochemical composition. Thus, the nucleus surrounded by cytoplasm with various densities and extracellular matrix were distinguished within intact undifferentiated and differentiated mouse ES cells which opens a great opportunity for Raman imaging in monitoring the stem cell differentiation on the early stages of embryogenesis. The major advantage of the non-invasive character of the technique is its capability to distinguish the biochemical environment within EB, since no stain is capable of penetrating the sample without the aid of membrane-permeabilizing protocols.