Using the MutSβ plus DNA assay format, a pilot screen of 17K compounds was conducted at both 20 µM and 40 µM compound concentration to assess the resulting hit-rate. Following hit confirmation, 315 compounds (1.8%) were identified from the 40 µM screen. These conditions were applied to a larger, diverse collection of 960K compounds and translated well, affording 21.7K (2.3%) primary hits at a 5σ threshold (7.1% median inhibition). Applying an additional filter of >20% inhibition reduced this number to approximately 10K compounds that could be advanced to hit confirmation.