This protocol describes a simple and an economical method for isolation of endothelial cells from human umbilical vein. Umbilical cord is easily available postpartum following informed consent, and the method for its collection is noninvasive with few ethical concerns. Thus, umbilical vein is an ideal source for isolation of endothelial cells of human origin. Endothelial cells are isolated from umbilical vein by collagenase digestion. This is followed by their culture on extracellular matrix (ECM) protein coated tissue culture flasks in presence of endothelial cell growth medium. In the last few decades, human umbilical vein endothelial cells (HUVECs) have come to be regarded as a standard model system by vascular biologists to understand general principles of endothelial cell biology and dysfunction. Here, we describe isolation, culture, as well as validation of HUVECs.