To begin our search for such small molecules we adapted the established ADP-Glo TM assay format. MutSβ was mixed with a heteroduplex 22bp (CAG)1-loop DNA to allow complex formation. Although basal MutSβ ATPase activity is detected in the absence of DNA, DNA substrates encoding a mismatch loop stimulate MutSβ ATPase activity (kcat)by 2-3 fold. A DNA loop substrate was included to improve assay signal and to ensure that the measured activity was DNAdependent. The MutSβ/DNA complex was then pre-incubated with test compounds, and ATP was added at its Km concentration (10 µM) and incubated for 60 minutes at 37 °C to allow hydrolysis of up to 10% of the ATP substrate. The ADP resulting from this reaction was then detected by addition of the ADP-Glo TM reagents, which stop the MutSβcatalyzed reaction, convert the remaining ATP into cAMP, and subsequently transform ADP back into ATP, which catalyzes luciferin luminescence. Using an ATP concentration equal to the MutSβ Km allowed the assay to be sensitive for both allosteric and orthosteric inhibitors, and this assay format was applied to screen a collection of small molecules.