These assays for cGMP, PGE2 and cortisol integrate proprietary d2 acceptor dye. The first assay quantifies cGMP (Cyclic Guanosine 3’, 5’-cyclic monophosphate), addressing the needs of screening targets such as guanylyl cyclases or a number of cGMP-regulated phosphodiesterases (PDEs); the second quantifies PGE2 (Prostaglandin E2), an essential inflammation mediator, either in cell supernatants or directly in the presence of whole cells; the third assay enables accurate cortisol measurement, even in complex samples such as liver microsomes, whole cells, and animal serum. The assays take advantage of the flexibility of homogeneous fluorescence: there is no separation, they are non-bead based and sensitive, and can be miniaturized down to 1536 format. Particularly well-adapted to and optimized for high-throughput screening (HTS), the assays’ procedures are rationalized to single-plate and “mix and measure” modes, can be adapted to cell-based format, and benefit from HTRF correction for media and compound interference.