Endothelial inflammation is the hallmark of vascular pathology often proceeding with cardiovascular diseases. Here, we adopted a multiparameter approach combining various imaging techniques at the nano- and microscale (Raman, AFM and fluorescence) to investigate endothelial inflammation in response to lipopolysaccharides (LPS) in vitro in human microvascular endothelial cells (HMEC-1) with a focus on lipid droplets (LDs) formation. Our results show that LPS-induced LDs in HMEC-1 have a composition depending on LPS-incubation time and their formation requires the presence of serum. Robust endothelial inflammation induced by LPS was linked to LDs composed of highly unsaturated lipids, as well as prostacyclin release. LPS-induced LDs were spatially associated with nanostructural changes in the cell membrane architecture. In summary, LDs formation represents an integral component of endothelial inflammation induced by LPS.