Gene silencing through RNA interference (RNAi) has become a powerful tool for understanding gene function. RNAi screens are primarily conducted using synthetic small interfering RNA (siRNA) or plasmid-encoded short hairpin RNA (shRNA). In this chapter, some considerations for design, optimization, validation, analysis and hit selection criteria in RNAi screens are discussed. A special emphasis is placed on pitfalls associated with off-target effects, which represent a primary limitation to the successful application of this technology.