The use of lentiviral vectors for transgene delivery in vitro and in vivo for applications in neuroscience, hematology, developmental biology, stem cell biology, and transgenesis has become commonplace. Lentiviral vectors provide an attractive tool for transgene delivery in part because of their ability to incorporate (integrate) into the genomic DNA of target cells with high efficiency, especially in cells that are not actively dividing. In addition, lentiviral vector-mediated transgene )expression can be maintained for long periods of time. In this unit, we describe protocols for lentiviral vector production in protein-free media using polyethylenimine (PEI)-mediated transfection, resulting in consistent lentiviral vector stocks. Such stocks are then concentrated by ultracentrifugation. We also provide reliable QPCR protocols to titrate lentiviral vectors based on vector DNA copies present in genomic DNA extracted from transduced cells. The vector production and titration protocol described here can be completed within 8 days.