Retroviral vectors have been used as gene delivery vehicles for more than two decades and continue to be the best available tool for stable and efficient transfer of therapeutic genes into various cell types. Although most gene therapy preclinical studies presently use crude or concentrated retroviral vector supernatants, purification to eliminate serum and host-derived impurities contained in these stocks will be a necessary requirement for clinical applications. Chromatography is deemed the most promising technology for large-scale purification of viral vectors. Heparin affinity chromatography offers the possibility to selectively and efficiently purify retroviruses. This chapter gives a simple, reproducible, and scaleable protocol for the purification of bioactive VSV-G pseudotyped retroviral vectors that employs membrane and chromatography technologies. The protocol can be easily adapted for the purification of different retroviral vector pseudotypes and lentiviral vectors. The purification techniques described here represent a significant improvement over the conventional sucrose density gradient methodology used for retrovirus purification and will hopefully contribute to the technological progress in the field of gene therapy.