Endothelial cells play a key role in prominent immunological and pathological processes such as leukocyte trafficking, inflammation, atheroma or cancer cell metastasis. Umbilical veins are probably the most widely used source for human endothelial cells, since they are more easily available than many other vessels, they are free from any pathological process and they are physiologically more relevant than many established cell lines. Here, we describe a standard protocol for preparation, maintenance and quality control of these cells.