A key characteristic of G protein-coupled receptors (GPCRs) is that they activate a plethora of signaling pathways. It is now clear that a GPCR coupling to these pathways can be selectively regulated by ligands that differentially drive signaling down one pathway in preference to another. This concept, termed stimulus bias, is revolutionizing receptor biology and drug discovery by providing a means of selectively targeting receptor signaling pathways that have therapeutic impact. Herein, we utilize a novel quantitative method that determines stimulus bias of synthetic GPCR ligands in a manner that nullifies the impact of both cellular background and the natural bias of the endogenous ligand. By applying this method to the M2 muscarinic acetylcholine receptor (mAChR), a prototypical GPCR, we found that mutation of key residues (Tyr802.61 and Trp993.28) in an allosteric binding pocket introduces stimulus bias in response to the atypical ligands AC-42 (4-n-butyl-1-(4-(2-methylphenyl)-4-oxo-1-butyl)piperidine HCl) and 77-LH-28-1 (1-(3-(4-butyl-1-piperidinyl)propyl)- 3,3-dihydro-2(1H)-quinolinone). By comparing stimulus bias factors between receptor internalization, G protein activation, extracellular-regulated protein kinase 1/2 (ERK1/2) signaling and receptor phosphorylation, we provide evidence that Tyr802.61 and Trp993.28 act as either as molecular switches or gatekeeper residues that introduce constraints limiting the active conformation of the M2 mAChR and thereby regulate stimulus bias. Furthermore, we provide evidence that downstream signaling pathways previously considered to be related to each other (i.e., receptor phosphorylation, internalization, and activation of ERK1/2) can act independently.