Prostaglandin E2 (PGE2) is a primary product of arachidonic metabolism and is synthesized via the cyclooxygenase (COX) and prostaglandin synthase pathways. PGE2 production is a commonly used method for the detection of COX-1 and COX-2 modulation and prostaglandin synthases. The HTRF® PGE2 assay is a highly sensitive method for quantifying PGE2 either in cell supernatants or directly in the presence of whole cells. Our reagents have simplified protocols requiring only two reagent additions. These protocols are easily miniaturized and amenable to automation and HTS.