The activation of heterotrimeric G proteins (Gαβγ) by G protein-coupled receptors (GPCRs) is a mechanism broadly used by eukaryotes to transduce signals across the plasma membrane and a target for many clinical drugs. Many optical biosensors commonly used for measuring GPCR-stimulated G protein activity rely on exogenously expressed GPCRs and/or G proteins, which compromise readout fidelity. Biosensors that measure endogenous signaling may interfere with the signaling process under investigation or have a limited dynamic range of detection, hindering applicability. Here, we developed an optical BRET-based biosensor, Gαi bONE-GO, that detects endogenous GTP-bound (active) Gαi upon stimulation of endogenous GPCRs more robustly than existing sensors of endogenous activity. Its design leverages the Gαi-binding protein GINIP as a high-affinity and specific detector of Gαi-GTP. We optimized this design to prevent interference with downstream Gi-dependent signaling and to enable implementation in different experimental systems having endogenous GPCRs, including adenosine receptors in primary astroglial cells and opioid receptors in cell lines. In a neuronal cell line, Gαi bONE-GO revealed activation profiles indicating that several natural opioid neuropeptides acted as partial agonists, in contrast with their characterization as full agonists using biosensors that depend on exogenously expressed receptors and G proteins. The Gαi bONE-GO biosensor is a direct and sensitive detector of endogenous activation of Gαi proteins by GPCRs in different experimental settings but does not interfere with the subsequent propagation of signaling.