Lentiviral vectors have evolved over the last decade as powerful, reliable, and safe tools for stable gene transfer in a wide variety of mammalian cells. Contrary to other vectors derived from oncoretroviruses, they allow for stable gene delivery into most nondividing primary cells. In particular, lentivectors (LVs) derived from HIV-1 have gradually evolved to display many desirable features aimed at increasing both their safety and their versatility. This is why lentiviral vectors are becoming the most useful and promising tools for genetic engineering, to generate cells that can be used for research, diagnosis, and therapy. This chapter describes protocols and guidelines, for production and titration of LVs, which can be implemented in a research laboratory setting, with an emphasis on standardization in order to improve transposability of results between laboratories. We also discuss latest designs in LV technology.