Many Food and Drug Administration (FDA)-approved drugs are structural analogues of the endogenous (natural) ligands of G protein-coupled receptors (GPCRs). However, it is becoming appreciated that chemically distinct ligands can bind to GPCRs in conformations that lead to different cellular signaling events, a phenomenon termed biased agonism. Despite this, the rigorous experimentation and analysis required to identify biased agonism are often not undertaken in most clinical candidates and go unrealized. Recently, xanomeline, a muscarinic acetylcholine receptor (mAChR) agonist, has entered phase III clinical trials for the treatment of schizophrenia. If successful, xanomeline will be the first novel FDA-approved antipsychotic drug in almost 50 years.Intriguingly, xanomeline's potential for biased agonism at the mAChRs and, in particular, the M 4 mAChR, the most promising receptor target for schizophrenia, has not been assessed. Here, we quantify the biased agonism profile of xanomeline and three other mAChR agonists in Chinese hamster ovary cells recombinantly expressing the M 4 mAChR. Agonist activity was examined across nine distinct signaling readouts, including the activation of five different G protein subtypes, ERK1/2 phosphorylation, β-arrestin recruitment, calcium mobilization, and cAMP regulation. Relative to acetylcholine (ACh), xanomeline was biased away from ERK1/ 2 phosphorylation and calcium mobilization compared to Gα i2 protein activation. These findings likely have important implications for our understanding of the therapeutic action of xanomeline and call for further investigation into the in vivo consequences of biased agonism in drugs targeting the M 4 mAChR for the treatment of schizophrenia.