MDM2 and XIAP are mutually regulated. Binding of MDM2 RING protein to the IRES region on XIAP mRNA results in MDM2 protein stabilization and enhanced XIAP translation. In this study, we developed a protein-RNA fluorescence polarization (FP) assay for high-throughput screening (HTS) of chemical libraries. Our FP-HTS identified eight inhibitors that blocked the MDM2 protein-XIAP RNA interaction, leading to MDM2 degradation. The compound-induced MDM2 downregulation resulted not only in inhibition of XIAP expression, but also in activation of p53, which contributed to cancer cell apoptosis in vitro and inhibition of cancer cell proliferation in vivo. Importantly, one of the MDM2/XIAP inhibitors, MX69, showed minimal inhibitory effect on normal human hematopoiesis in vitro and was very well tolerated in animal models. Display omitted •MDM2/XIAP inhibitors are selected by FP-HTS via blocking MDM2-XIAP IRES interaction•MDM2/XIAP inhibitors degrade MDM2, activate p53, and inhibit XIAP expression•MDM2/XIAP inhibitors exhibit anticancer activity in vitro and in animal models•One of the MDM2/XIAP inhibitors MX69 is very well tolerated in animals Gu et al. use chemical screening to identify inhibitors of the MDM2 protein-XIAP mRNA interaction and show these inhibitors lead to apoptosis by reducing XIAP protein, decreasing MDM2 stability, and increasing p53 levels. Animal testing of one of these compounds shows antitumor efficacy and minimal toxicity.