Abstract E3 ubiquitin ligases are of interest as drug targets for their ability to regulate protein stability and function. The oncogene Mdm2 is an attractive E3 ligase to target, as it is the key negative regulator of the tumor suppressor p53, which controls the transcription of genes involved in cell fate. Overexpression of Mdm2 facilitates tumorigenesis by inactivating p53, and through p53-independent oncogenic effects. We developed a high-throughput cellular Mdm2 auto-ubiquitination assay, which we used to discover a class of small-molecule Mdm2 ligase activity inhibitors. These compounds inhibit Mdm2 and p53 ubiquitination in cells, reduce viability of cells with wild-type p53, and synergize with DNA-damaging agents to cause cell death. We determined that these compounds effectively inhibit the E3 ligase activity of the Mdm2-MdmX hetero-complex. This mechanism may be exploitable to create a new class of anti-tumor agents. Significance: We identified a class of small-molecule inhibitors of the Mdm2-MdmX hetero-complex E3 ligase activity through a high-throughput cell-based Mdm2 ubiquitination screen. This is a new target for small-molecule therapeutics and may be developed to treat specific cancers. Cancer Discovery; 1(4); 312–25. ©2011 AACR. This article is highlighted in the In This Issue feature, p. 275