Guanosine triphosphate binding protein (G protein)-coupled receptors (GPCRs) are a large class of pharmaceutical drug targets. With the increasing popularity of functional assays for high throughput screening, there arises an increasing need for robust second messenger assays that reflect GPCR activation and are readily amenable for miniaturization. GPCRs that upon agonist stimulation modulate adenylyl cyclase activity, and, consequently, cellular cyclic adenosine monophosphate (cAMP) levels, via the G protein Gs or Gi, form a subset of therapeutic targets. While there are several cAMP assays currently available, most are not scalable for miniaturization into the 1536-well format employed for automated high throughput screening of large chemical libraries. Here, we describe a cAMP assay based on the enzyme fragmentation complementation (EFC) of beta-galactosidase. In this assay, recombinant cells expressing Gs- or Gi-coupled receptors exhibit robust and reproducible pharmacology for agonists and antagonists, as measured by cAMP levels. Furthermore, the EFC cAMP assay offers sufficient sensitivity to be used with cells expressing endogenous GPCRs. We demonstrate the miniaturization of this assay into a 1536-well format with comparable sensitivity and plate statistics to those of the 384-well assay for both Gs- and Gi-coupled receptors, and its suitability for miniaturized high throughput screening.