Since it was found that synthetic small interfering RNA (siRNA) can invoke RNA interference (RNAi) responses in mammalian cells, it has gained enormous attention as a tool for gene silencing in basic science and as a novel therapeutic modality. To develop carriers for cytosolic and systemic siRNA delivery, our laboratory has recently developed a sequence-defined polymer platform compatible with solid-phase-supported synthesis. These polymers have displayed efficient siRNA-mediated gene silencing in vitro and in vivo. In this chapter, we provide a brief background on the special features of these polymers and detailed protocols to evaluate polyplex formation, gene silencing efficiency, and cytotoxicity of siRNA-containing polyplexes.