Lentiviral (LV) vectors offer unique advantages over other gene delivery systems, namely the ability to integrate transgenes into the genome of both dividing and nondividing cells. Detailed herein is a simple protocol for the production LV vectors, describing the triple transfection of an LV transfer vector and LV helper plasmids into HEK-293 cells, and the subsequent purification of virions from the cellular media. The current protocol is versatile, and can be easily modified to fit the specific needs of the researcher in order to produce relatively high-titer LV vectors which can be used to transduce a wide variety of cells both in vitro and in vivo.