[Show abstract][Hide abstract] ABSTRACT: Reducing costs while maintaining the highest readout quality is a precept of modern high-throughput screening. Given the trend toward nonradiometric screening platforms, this has been a big challenge for some kinase target classes. Common issues include low sensitivity, susceptibility to nonspecific interference, or the need for costly reagents. In this study, the authors describe the feasibility of miniaturization of a serine kinase assay using generic reagents in the AlphaScreen format. They have validated the robustness of this assay in the course of miniaturization from a 35-to 4.375-microL final assay volume in 384-and 1536-well formats. Within this volume range, they consistently obtained Z' values above 0.5 and have investigated the suitability of these assay formats for measuring compound effects by testing a set of 25 previously identified active compounds. These active compounds were also reliably identified in the miniaturized assay formats. The results presented here show that the AlphaScreen technology permits robust and cost-efficient miniaturization of serine/threonine kinase assays.