Cancer is a devastating disease characterized by uncontrolled and aggressive cell growth. Suicide gene therapy (SGT) facilitating induction of malignancy-specific cell death represents a novel therapeutic approach to treat cancer, which has been investigated in several cancer types with very promising results. In addition, SGT has been suggested as a safeguard in adoptive immunotherapy and regenerative-medicine settings. Generally, SGT consists of two steps-vector-mediated delivery of suicide genes into tumors and subsequent activation of the suicide mechanism, e.g., by administration of a specific prodrug. This chapter provides a framework of protocols for basic and translational research using the Herpes-simplex-virus thymidine kinase (HSV-TK)/ganciclovir (GCV) system, the most widely used suicide gene approach. The protocols provide standard guidelines for the preparation of high-titer third-generation lentiviral vectors encoding a genetically improved HSV-TK version known as TK.007 and its application in in vitro and in vivo treatment setups.