Ligand-activated, seven transmembrane-spanning receptors interact with inactive G-protein heterotrimers (Gαβγ) to catalyze GTP loading and, consequently, activation of Gα subunits and the liberation of Gβγ. Gα·GTP and Gβγ are then competent to regulate independent effector pathways. The duration of heterotrimeric G-protein signaling is determined by the lifetime of the Gα subunit in the GTP-bound state. Signal termination is facilitated by the intrinsic guanosine triphosphatase (GTPase) activity of Gα and subsequent reformation of the inactive heterotrimer. Regulators of G-protein signaling (RGS) proteins act enzymatically, via their hallmark "RGS box," as GTPase-accelerating proteins (GAPs) for Gα subunits and thus function as negative regulators of G-protein signaling in vitro and in vivo. This article describes the use of fluorescence resonance energy transfer (FRET) to monitor the interaction between a Gα subunit and an RGS box protein. Furthermore, this article describes optimization of this assay for high-throughput screening and the evaluation of mutant RGS box and Gα proteins. Finally, this article describes the novel application of this FRET technique to measure the activity of RGS protein-derived GoLoco peptides that modulate Gα activation by aluminum tetrafluoride.