3',5'-Cyclic adenosine monophosphate (cAMP) is a common intracellular second messenger that enables cells to respond to external stimuli. Measurement of intracellular cAMP concentrations is thus widely used for studying guanosine triphosphate binding protein-coupled receptors (GPCRs), which make up a large class of pharmaceutical drug targets. Although several assay technologies exist to measure cAMP, most are not suitable for ultra-high-throughput screening (uHTS), as is often required for screening large (greater than 1 million) chemical libraries for the identification of suitable leads for drug development. Here we report that the enzyme fragment complementation assay, a homogeneous gain of signal assay based on complementation of two fragments of a beta-galactosidase enzyme, is compatible with uHTS requirements of a 2.2-microl total assay volume in 3,456-well plate format. We describe the miniaturization of this assay into 3,456-well plate format exhibiting comparable sensitivity and plate statistics to those of a 384-well assay and the application of this assay in uHTS for the identification of antagonists of a Gi-coupled receptor.