In sum, we describe and validate a phenotypic screening strategy to identify small molecules that enhance the activity of the type I IFN signaling pathway and consequently improve antiviral host defense. This approach is designed to lead to discovery of drugs with activity against a broad range of viruses for clinical application as well as experimental tool compounds to further understand IFN-dependent immune mechanisms. Current approaches to defining the basis for IFN signal transduction, particularly in vivo, often rely on complex transgenic and gene targeting approaches. Thus, the use of small molecule enhancers (SMEs) of the IFN signaling pathway may provide much greater flexibility and ease of application to achieve transient adjustment of IFN-related actions and consequent scientific and clinical benefit. Our approach should thereby prove useful to discover drugs with activity against a broad range of viruses as well as effectiveness in other conditions (e.g., multiple sclerosis and melanoma) where the efficacy of IFN treatment might benefit from enhancing the IFN signaling pathway.