We sought to develop a small-molecule activator of interferon regulatory factor 3 (IRF3), an essential innate immune transcription factor, which could potentially be used therapeutically in multiple disease settings. Using a high-throughput screen, we identified small-molecule entities that activate a type I interferon response, with minimal off-target NFκB activation. We identified 399 compounds at a hit rate of 0.24% from singlicate primary screening. Secondary screening included the primary hits and additional compounds with similar chemical structures obtained from other library sources and resulted in 142 candidate compounds. The hit compounds were sorted and ranked to identify compound groups with activity in both human and mouse backgrounds to facilitate animal model engagement for translational development. Chemical modifications within two groups of small molecules produced leads with improved activity over original hits. Furthermore, these leads demonstrated activity in ex vivo cytokine release assays from human blood- and mouse bone marrow-derived macrophages. Dependence on IRF3 was demonstrated using bone marrow-derived macrophages from IRF3-deficient mice, which were not responsive to the molecules. To identify the upstream pathway leading to IRF3 activation, we used a library of CRISPR knockout cell lines to test the key innate immune adaptor and receptor molecules. These studies indicated a surprising toll-interleukin-1 receptor-domain-containing-adapter-inducing interferon-β-dependent but TLR3/4-independent mechanism of IRF3 activation.