Biased agonism at G protein coupled receptors emerges as an opportunity for development of drugs with enhanced benefit/risk balance making biased ligand identification a priority. However, ligand biased signature, classically inferred from ligand activity across multiple pathways, displays high variability in recombinant systems. Functional assays usually necessity receptor/effector overexpression that should be controlled among assays to allow comparison but this calibration currently fails. Herein, we demonstrate that Gα expression level dictates the biased profiling of agonists and, to a lesser extent of β-blockers, in a Gα isoform- and receptor-specific way, depending on specific G protein activity in different membrane territories. These results have major therapeutic implications since they suggest that the ligand bias phenotype is not necessarily maintained in pathological cell background characterized by fluctuations in G protein expression. Thus, we recommend implementation of G protein stoichiometry as a new parameter in biased ligand screening programs.