It is clear that viral entry, replication, and spread is a complex process involving a dialog between the virus and the targeted host cell. Viruses have evolved highly specific strategies to hijack cellular factors to promote their internalization, initiate their replication, and facilitate their eventual spread. However, the identification of many of these host cell molecules has been hindered by the requirement for robust genome-scale loss-of-function assays that are capable of targeting a wide variety of host factors. The more recent use of genome-scale or genome-wide RNA interference (RNAi) screens have extended our knowledge of the complex interplay between a virus and host and have implicated a wide variety of cellular factors required for infection of a number of viruses. Here, we describe an approach to target mammalian host cell factors involved in regulating viral infections by the use of a genome-scale RNAi library screen.