Considered by the WHO as the world’s most disabling psychiatric disorder, major depressive disorder (MDD) severely affects patients’ quality of life. Treating patients with serotonin reuptake inhibitors (SSRIs), the first-line treatment, improve symptoms but have many limitations, such as delayed clinical benefit and resistance to treatment. Recently, clinical evidences showed that hallucinogenic agonists of the 5-HT2A serotonin receptor, such as LSD or psilocybin, induce rapid and lasting antidepressant effects. 5-HT2A receptor activation is known to promote synaptic plasticity, but the molecular mechanisms underlying the antidepressant effects of its agonists still remain to be elucidated. Deciphering 5-HT2A downstream targets would provide insights to identify new actors involved in the antidepressant effects of hallucinogenic drugs. Phosphorylation events are key modulators of GPCRs signaling and play a central role in regulating synaptic plasticity. Thus, we investigated synaptic phosphoproteome induced upon 5-HT2A receptor stimulation by the DOI, a hallucinogenic agonist, in the prefrontal cortex of wild-type and htr2A-/- mice. We identified 14 phosphorylation sites specifically induced by 5-HT2A receptor’s activation. Among the phosphorylated identified proteins, several are involved in metabotropic glutamate receptor 5 (mGLUR5) signalosome. Using primary cultures of cortical neurons, we demonstrated a functional interaction between the mGlu5 and 5-HT2A receptors. This work provides a better understanding of molecular mechanisms induced upon 5-HT2A activation by agonist with hallucinogenic property.