A single-format method to detect multiple G protein–coupled receptor (GPCR) signaling, especially Gα_12/13 signaling, presently has limited throughput and sensitivity. Here we report a transforming growth factor-α (TGFα) shedding assay, in which GPCR activation is measured as ectodomain shedding of a membrane-bound proform of alkaline phosphatase–tagged TGFα (AP-TGFα) and its release into conditioned medium. AP-TGFα shedding response occurred almost exclusively downstream of Gα_12/13 and Gα_q signaling. Relying on chimeric Gα proteins and promiscuous Gα₁₆ protein, which can couple with Gα_s- and Gα_i-coupled GPCRs and induce Gα_q signaling, we used the TGFα shedding assay to detect 104 GPCRs among 116 human GPCRs. We identified three orphan GPCRs (P2Y10, A630033H20 and GPR174) as Gα_12/13-coupled lysophosphatidylserine receptors. Thus, the TGFα shedding assay is useful for studies of poorly characterized Gα_12/13-coupled GPCRs and is a versatile platform for detecting GPCR activation including searching for ligands of orphan GPCRs.