Modern drug discovery has embraced in vitro platforms that enable investigation of large numbers of compounds within tractable timeframes and for feasible costs. These endeavors have been greatly aided in recent years by advances in molecular and cell-based methods such as gene delivery and editing technology, advanced imaging, robotics, and quantitative analysis. As such, the examination of phenotypic impacts of novel molecules may only be limited by the size of the compound collection. Innate immune signaling processes in mammalian cells are especially amenable to high-throughput screening platforms since the cellular responses elicited by their activation often result in high level transcription that can be harnessed in the form of bioluminescent or fluorescent signal. In addition, targeted activation of innate immune pathways represents a valuable therapeutic strategy applicable to multiple chronic and acute human diseases. Herein, we describe the optimization and utilization of a high-throughput screening method using human reporter cells reactive to stimulation of the type I interferon response. Importantly, the principles and methods described can be applied to adherent reporter cells of diverse derivation and innate signaling pathway readouts.