AbstractExtensive functional studies of the exchange protein directly activated by cAMP (EPAC) family of signaling molecules have demonstrated that EPAC proteins play a fundamental role in several physiological and pathophysiological responses, therefore are attractive drug targets. In this report, the development of a cell-based, medium to high throughput screening assay that is capable of monitoring EPAC-mediated activation of cellular Rap1 in an isoform-specific manner is described. This assay adapts a conventional ELISA format with immobilized RalGDS-RBD as a bait to selectively capture GTP-bound active Rap1. As a result, it fills an urgent need for a cell-based EPAC assay that can be conveniently performed using microtiter plates for the discovery and/or validation of isoform-specific EPAC agonists and antagonists.