G protein coupled receptors (GPCRs) are one of the largest receptor classes targeted by drug discovery programs. Calcium flux (coupled via Gq pathway) assay is a preferred method in drug discovery for screening GPCR targets. However, over 60% of the known GPCRs signal through adenylyl cyclase activity coupled to cAMP. Most of the existing cAMP assays not only require cell lysis but also lack both temporal and spatial resolution. Screen Quest™ cell lines enable to investigate GPCRs that do not conventionally couple through intracellular calcium. Screen Quest™ cell lines are based on a series of G-protein chimeras, including the promiscuous G-protein, Gα16, and an exogenous cyclic nucleotide-gated channel (CNGC). The chimeras consist of the alpha subunit of a Gq-protein complex whose 5 carboxy-terminal amino acids have been replaced with those from one of the other G-proteins (either Gαs, Gαi, Gαo, or Gαz). These amino acids are responsible for the coupling of the receptor to its G-protein. Co-expression of these chimeras or CNGC with specific non-Gq-coupled receptors may result in the generation of an intracellular calcium signal upon receptor stimulation. Screen Quest™ HEK-CNGC-Opiate Receptor-like 1 (ORL1) cell line is HEK-293 cells stably transfected with both the CNGC and human Opiate Receptor-like 1. The constitutively expressed CNGC in the cells responds in real-time to increases or decreases in intracellular cAMP levels by coordinately altering cation flux (e.g., calcium, potassium or sodium). Activation of the ORL1 in these cells by specific ligands can be detected with either a calcium-sensitive fluorescent indicator (such as Calbryte 520 AM, Cal-520® AM, Fluo-8® AM, or Fluo-4 AM and the corresponding no wash calcium kit) or an AAT’s optimized membrane-potential assay kit. This cell line has been successfully used in drug discovery and screening environments for studying GPCRs that do not conventionally couple through intracellular calcium. It has been effectively used with the FLIPR and FDSS systems.