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Potent and highly selective small-molecule inhibitors are needed to unravel the biological complexities of histone methyltransferases and to reveal their therapeutic potential. A prerequisite to developing these inhibitors is the identification of validated chemical matter for initiating a medicinal chemistry campaign. For the most part, finding these initial starting points occurs through screening of large, unbiased compound libraries. The size and nature of these libraries, coupled with the complexities of the bisubstrate utilizing histone methyltransferases, necessitates that the primary screen and subsequent hit triage be carefully considered.In this chapter, using EZH2 as a representative example, we describe a screening and hit triage campaign that identified validated chemical matter allowing initiation of medicinal chemistry studies. Moreover, we discuss a cell-based assay to support lead identification and optimization. The approach described here entailing a mixture of biochemical, biophysical and cell-based assays should be applicable to identifying validated starting points for other histone methyltransferases., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)