RNA interference (RNAi) is a cellular mechanism that mediates sequence-specific gene silencing by cleavage or translational inhibition of the targeted mRNA. RNAi can be used as an antiviral approach to silence the human immunodeficiency virus type 1 (HIV-1). The first clinical trial using RNAi against HIV-1 in a lentiviral gene therapy setting was initiated in early 2008. In this chapter, we will focus on the basic principles of such an RNAi-based gene therapy against HIV-1. Subjects that will be covered include target site selection within the viral RNA genome, viral escape, and therapeutic strategies to prevent this, such as combinatorial RNAi approaches, systems available for multiplexing of RNAi inhibitors, methods to deliver the antiviral RNAi molecules and gene therapy protocols to achieve durable HIV-1 inhibition. We will also discuss several in vitro and in vivo test systems to evaluate the efficacy and safety of an RNAi gene therapy.