Although only 16 genes have been identified in mammals, several G-subunits can be simultaneously activated by G protein-coupled receptors (GPCRs) to modulate their complicated functions. Current GPCR assays are limited in the evaluation of selective G activation, thus not allowing a comprehensive pathway screening. Because adenylyl cyclases are directly activated by G_s and the carboxyl termini of the various G proteins determine their receptor coupling specificity, we therefore proposed a set of chimeric G_s where the C-terminal five amino acids are replaced by those of other G proteins and used these to dissect the potential G linked to a given GPCR. Unlike G_q, G₁₂ and G_i outputs, compounding the signals from several G members, the chimeric G_s proteins provide a superior molecular approach that reflects the previous uncharacterized pathways of GPCRs under the same cAMP platform. This is, to our knowledge, the first time allowing verification of the whole spectrum of G-coupling preference of adenosine A1 receptor, reported to couple to multiple G proteins and modulate many physiological processes. Further, we were able to distinguish the uncharacterized pathways between the two neuromedin U receptors (NMURs), which distribute differently but are stimulated by a common agonist. In contrast to the G_q signals mainly conducted by NMUR1, NMUR2 routed preferentially to the G_i pathways. Dissecting the potential G coupling to these GPCRs will promote an understanding of their physiological roles and benefit the pharmaceutical development of agonists/antagonists by exploiting the selective affinity towards a certain G subclass.