RNA interference is a gene silencing phenomenon triggered by dsRNA (double-stranded RNA) and has been widely used for studying gene functions. The short interfering RNA (siRNA) responsible for RNA interference varies markedly in its gene silencing efficacy. This book reviews the recently reported siRNA design guidelines and clarifies the problems concerning the guidelines. An effective method for selecting siRNA target sequences from many possible candidate sequence is also described by using Bayes’ theorem. Furthermore, the development of a durable RNAi therapy for cancer and viral infections, which may require the use of multiple inhibitors is evaluated. This book also reviews the structure, application and therapeutic challenges of siRNA.