Circulating endothelial cells (CECs) are shed from the vascular intima into the bloodstream during endothelial injury and serve as a minimally invasive indicator of vascular dysfunction in sepsis. In this study, we isolated CECs from peripheral blood samples of patients with sepsis and healthy donors using high-dimensional spectral flow cytometry (Hoechst⁺/7-AAD⁻/CD45⁻/CD34⁺/CD146⁺). Due to the extremely low abundance of CECs in circulation, a nanoproteomics workflow optimized for trace cellular protein profiling was applied.