RNA interference (RNAi) is a naturally occurring cellular defense mechanism against viral infections and transposon invasion. Short double-stranded RNA molecules, so-called small-interfering (si)RNAs, bind their complementary mRNA leading to the mRNA's degradation. During the past few years, RNAi has become a valuable tool for transient as well as stable repression of gene expression rendering the time-consuming production of knockout animals superfluous. In this chapter the usability of the RNAi technology in cancer research will be described, focusing on the application of large-scale screens for identification of new components in cancer-relevant signal pathways (e.g., p53, RAS). The screens are especially helpful in the detection of potential anticancer drug targets or siRNAs with therapeutic potential.