The most widely used approach for defining gene function is to reduce or completely disrupt its normal expression. For over a decade, RNAi has ruled the lab, offering a magic bullet to disrupt gene expression in many organisms. However, new biotechnological tools—specifically CRISPR-based technologies—have become available and are squeezing out RNAi dominance in mammalian cell studies. These seemingly competing technologies leave research investigators with the question: “Which technology should I use in my experiment?” This review offers a practical resource to compare and contrast these technologies, guiding the investigator when and where to use this fantastic array of powerful tools. Boettcher and McManus provide a practical guide to choosing the right tool for your gene silencing experiment—RNAi, TALEN, or CRISPR.