Type I interferons (IFN-α/β) have been widely used in the treatment of many viral and malignant diseases by activation of the Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling pathway, but the side effects of protein-based IFN therapy severely limit their clinical use. Discovering small molecules to activate the JAK/STAT pathway will greatly facilitate the development of new drugs which have similar pharmacological function to IFNs but with fewer side effects. To screen a natural products-based library, we established a cell-based screening assay using human hepatoma HepG2 cells stably transfected with a plasmid where the luciferase reporter activity is driven by interferon α-stimulated response element (ISRE), the motif specifically recognized by type I IFN-induced activation of JAK/STAT pathway. Among 1,431 natural product compounds screened, four compounds (emodin, quercetin, apigenin and luteolin) were identified as activators of the JAK/STAT pathway. Further studies demonstrated that these four compounds could increase the endogenous antiviral gene expression regulated by the IFN-activated JAK/STAT pathway. The identified small molecule activators are valuable for structural modification and warrant further investigation for use in new antiviral drugs as IFN mimics or adjuvants.