Despite great achievements in cancer chemotherapy, restricting the toxic effects of chemotherapeutic agents to tumor cells in order to enhance efficacy and reduce side effects remains a major challenge. To overcome this challenge, suicide gene therapy or gene-directed enzyme prodrug therapy (GDEPT), which is an alternative two-step process to conventional cancer treatment methods, is investigated. A gene that expresses a nontoxic enzyme in cancer cells is first delivered to cancer cells, followed by systemic administration of a prodrug that can be converted into a toxic compound by the enzyme. Factors that could impact the success of GDEPT include the level of enzyme expression at the target site, the efficiency of prodrug conversion into toxic drug, and the ability of drug to diffuse into neighboring cells. This chapter describes some of the most widely used enzyme/prodrug systems in preclinical and clinical trials along with various methods of cell targeting for GDEPT.