The development of HTS in the pharmaceutical industry has resulted in calcium assay becoming the most widely utilized for the characterization of receptors ligands interactions within whole cells. Due to the rapid and complex nature of calcium signal, the system is unlikely to reflect equilibrium binding conditions. Thus, there are potential issues associated with the interpretation of drug potency when performing SAR for GPCRs. Schild regression analyses are arguably the most appropriate method to determine antagonist potency from functional experiments. pA2 is used as a universal determinant of antagonist potency to overcome the potential bias associated with non- equilibrium conditions. It is then possible to estimate the affinity of insurmountable antagonists by calculating pA2 from dose ratios at low agonist responses. Additional experimental parameters should be taken into account when performing calcium assays. The diffusion properties for agonists appeared as important parameters to be considered during evaluation of antagonist compounds. Precise and defined agonist diffusion parameters have to be closely determined for any given GPCR when implementing calcium assays. Here we focus on studying depth and rate of agonist injection.