Excess circulating fatty acids contribute to endothelial dysfunction that subsequently aggravates the metabolic conditions such as fatty liver diseases. However, the exact mechanism of this event is not fully understood, and the investigation on the effect of a direct exposure to fatty acids together with their subsequent fate is of interest. In this work we employed a chemically specific and label-free techniques such as Raman and CARS microscopies, to investigate the process of lipid droplets (LDs) formation in endothelial cells and hepatocytes after exposure to oleic and palmitic acid. We aimed to observe the changes in the composition of LDs associated with metabolism and degradation of lipids. We were able to characterize the diversity in the formation of LDs in endothelium as compared to hepatocytes, as well as the differences in the formation of LDs and degradation manner with respect to the used fatty acid. Thus, for the first time the spectral characteristics of LDs formed in endothelial cells after incubation with oleic and palmitic acid is presented, including the time-dependent changes in their chemical composition.