Liver sinusoidal endothelial cells (LSECs) represent a highly specialized and unique type of endothelial cell in terms of their morphology and function. The biochemical and functional characterization of LSECs in vitro is restrained by the rapid change of LSECs' phenotype upon culturing under classical experimental conditions. In this work, we present a novel approach to characterize the biochemical content of murine LSECs, freshly isolated from the liver, with the use of microspectroscopic analysis. For comparison, hepatocytes and Hepatic Stellate Cells (HSCs) were analyzed. Our approach, based on label-free confocal Raman imaging of live cells combined with chemometric analysis, provided insight into the biochemical content of freshly isolated LSECs on a subcellular level. LSECs were featured by a distinct biochemical signature in comparison with other major cell types of the liver. Based on our work we claim that the non-invasive and non-destructive confocal Raman imaging may assist in obtaining chemical information spatially distributed within the cells that characterize the phenotype of primary LSECs as well as other types of liver cells. Furthermore, our approach provides a unique insight into LSECs' morphology and chemical composition that may help to understand their functions.