G-Protein Coupled Receptors (GPCRs) have been an important target class due to their involvement in a plethora of physiological processes. Their high druggability has led to the discovery of many medicines used to treat various diseases and conditions by modulating their functions. Understanding of the molecular mechanisms of GPCR functions has been advancing through the years. This knowledge opens up new opportunities to modulate this family of drug targets beyond the traditional agonists and antagonists, to include allosteric modulators and biased ligands. Benefiting from this new knowledge necessitates updates in the methodologies used for the drug discovery process, especially during the lead discovery and optimization phases. This chapter, based on current understanding of GPCR functions, describes various important mechanistic pharmacology assays useful during lead discovery and optimization. These assays can be applied to better understand the underlying pharmacological properties of compounds and increase the chance of success.