Primary cells are a valuable tool for researchers and are often preferred over transformed or immortalized cell lines since they are biologically more relevant and resemble the in vivo situation much closer. Unfortunately, efficient gene transfer in primary cells is still limited. Whereas viral strategies are time consuming and involve safety risks, nonviral methods are often inefficient for most primary cells. Nucleofection has been proven to overcome these limitations. Here, we describe the Nucleofection protocol for efficient transfection of human umbilical vein endothelial cells. Using a combination of a cell type-specific solution and electrical conditions, transfection efficiencies up to 90% can be achieved while survival rate is more than 70%.