ONE vector G protein optical (ONE-GO) biosensors are versatile tools to measure the activity of G protein-coupled receptors (GPCRs) in cells. The availability of ONE-GO biosensors for ten active Gα subunits representative of all four G protein families (Gs, Gi/o, Gq/11, and G12/13) permits the study of virtually any GPCR. Here, we present a protocol to implement ONE-GO biosensors in cell lines to investigate GPCR signaling kinetics and concentration-dependent responses. We describe steps for cell culture and transfection, response measurement, and data analysis. For complete details on the use and execution of this protocol, please refer to Janicot et al.1.