This chapter discusses guanosine 5'-O-(γ-Thio)triphosphate (GTP) binding assay for solubilized G proteins. Receptor activation of trimeric (αβγ) G proteins involves the release of bound GDP and the binding of GTP to the α subunit. When a receptor coupled to a G protein binds agonist, the receptor changes conformation in such a way as to promote this exchange. The nucleotide exchange results in activation of the a subunit and its dissociation from the βγ subunits. The α subunit has GTPase activity and will eventually hydrolyze bound GTP to GDP and thus return to its inactive state. It is only able to affect its target enzyme or channel during that time period when GTP is bound but not yet hydrolyzed. If a nonhydrolyzable analog of GTP is bound, the α subunit becomes persistently activated. Guanosine 5'-O-(γ,-thio)triphosphate (GTPγS) is such an analog. G proteins binds GTPγS rapidly and irreversibly even in the absence of receptors. The conditions required for optimal binding of GTPγS to these G proteins is well defined in the chapter.