We have developed novel strategies and powerful libraries for rapidly creating and conducting screens in mammalian cells.Existing short hairpin RNA libraries are greatly limited by low efficacy of many shRNAs and by off-target effects, which give rise to false negatives and false positives, respectively.Our systems center on EXPANDed RNAi technology, where ultra complex pools of shRNAs (~30 per gene) are analyzed by deep sequencing.This approach enables the identification of multiple effective target-specific shRNAs from complex pools, allowing a rigorous statistical evaluation of true hits.We have used this technology in a variety of different experimental contexts, significantly improving the art of pooled screens in mammalian cells, and laying a critical foundation for drug target discovery.