RNA interference (RNAi) is a powerful tool for the functional analysis of essential genes in the mammalian genome. Here, we present a simple ecdysone-based inducible RNAi approach that allows high induction and adjustable control of short hairpin RNA (shRNA) expression for silencing gene expression in a wide range of mammalian cells. This protocol describes the following: the design and cloning of inducible shRNA; testing and validation of gene knockdown; and methodology for establishing stable cell lines. This step-by-step protocol offers a quick and cost-effective approach for addressing the function of genes essential for cell cycle regulation and development and can be completed in less than 6 weeks.