Our primary screen identified idarubicin on the basis of its capacity to significantly increase ISRE activity. Subsequent validation assays demonstrated that idarubicin facilitates STAT1-dependent ISG expression and STAT1-directed control of viral replication and cytopathic effect. While others previously reported the antiviral properties of anthracyclines some time ago, no mechanism of their antiviral action was elucidated [46] [47] [48] . In the present study, we observed drug-induced cytotoxicity in a dose-range similar to those reported previously [23, 24] , however, we establish that the effect of idarubicin on the antiviral IFN pathway is independent of cytotoxicity and topoisomerase inhibition. Because idarubicin enhances the IFN signaling pathway output, we questioned whether the drug might also cause IFN-driven cell death. However, we found no increase in cytotoxicity in cells treated with idarubicin and IFN together compared to cells treated with idarubicin alone. We also found that the idarubicin concentrations for activating the ISRE component of the IFN signaling pathway were significantly less than those required for major cytotoxicity. Thus, we conclude that idarubicin effect on IFN signaling is distinct from the effect on DNA-based cytotoxicity. The dose dependency of these effects underlines the need to conduct screening at multiple concentrations of test compounds, particularly lower concentrations that prevent false negative hits due to cytotoxic effects.