Lytic induction of latent Kaposi's sarcoma-associated herpesvirus (KSHV) has been considered as a therapeutic option for efficient treatment of several KSHV-associated malignancies. Here, we developed a robust high-throughput screening system that allows an easy and quantitative measurement of lytic induction of latent KSHV and discovered three anthracyclines as potent inducers from screen of FDA-approved drugs. Lytic induction of latent KSHV by three compounds was verified by the significant induction of lytic genes and subsequent production of infectious KSHV. Importantly, lytic induction by three compounds was much more efficient than that by sodium butyrate, a well-characterized inducer of KSHV lytic cycle. Mechanistically, the anthracyclines caused lytic induction of KSHV through apoptosis induced by their DNA intercalation rather than topoisomerase II inhibition. Consequently, our results clearly demonstrated a role of anthracyclines as effective lytic inducers of KSHV and also provided a molecular basis of their use for efficient treatment of diseases associated with KSHV infection.