Unsatisfied with the quality of chemical matter remaining and concerned by the high attrition observed during hit triage, we revisited our primary assay with the intent of providing more stringent conditions for the removal of artefacts. The use of a decoy protein to minimize aggregationbased artefacts has been previously reported. Following additional assay optimization, a second version of the ADP-Glo TM assay was implemented, utilizing (i) ovalbumin as a decoy protein, (ii) changing the order of reagent addition where enzyme is added after substrate, (iii) higher concentrations of a different detergent and (iv) DTT was replaced by GSH. An orthogonal readout assay was also established, where hydrolyzed Pi is absorbed non-specifically to SPA beads after formation of a hydrophobic molybdate complex. The SPA assay was conducted in a decoy-free buffer to avoid readout interference.