E3 ubiquitin ligases are a large family of proteins that, together with ubiquitin-activating enzyme E1 and ubiquitin-conjugating enzyme E2, catalyze the ubiquitination of a variety of protein substrates for targeted degradation by means of the 26S proteasome. Because the turnover of many proteins involves targeted ubiquitination and degradation, E3 ubiquitin ligases, therefore, regulate almost every aspect of eukaryotic cellular functions or biological processes. Accumulated evidence in the past few years has suggested that a subset of E3 ubiquitin ligases that regulates the turnover of tumor suppressors and cell cycle regulators could be promising targets for mechanism-driven cancer drug discovery. Thus, it is highly desirable to optimize the methods of high-throughput screening (HTS) for specific inhibitors of these E3 ubiquitin ligases. Here I will give an overview of several approaches used for HTS for ubiquitin ligase inhibitors with a main focus on assay principles, applications, and the pros and cons of each approach. Experimental details for many of these assays can be found in other chapters in this volume.