Signal transduction via G-protein coupled receptors (GPCRs) is mediated through binding of GTP to GTP‑binding proteins. Evaluation of GPCR agonists in drug discovery requires GTP binding assays to confirm agonist activity. This chapter addresses the development and optimization of robust GTPγS binding assays with discussions on basic protocols, assay buffers, effect of required additives (Mg2+, NaCl, Saponin) and data analysis. An extensive set of recent references is included for beginners and experienced investigators.