Significance G protein–coupled receptors (GPCRs) have been proposed to signal in multiple compartments within the cell, a phenomenon that could have important implications for our understanding of receptor pharmacology and disease treatment. However, directly measuring G protein activity in subcellular compartments remains a challenge, and the mechanism underlying their translocation and activation remains elusive. Using a biosensor that specifically measures the presence of GTP-loaded G _q/11 at endosomes, our findings reveal the mechanism underlying G _q trafficking and signaling in the endosome and provide a powerful tool to directly monitor G protein movement and activation in subcellular compartments.