Psilocin is the active metabolite of psilocybin, a serotonergic psychedelic substance. It is used recreationally and investigated in substance-assisted psychotherapy. The pharmacokinetic properties of psilocin are only partially characterized. Therefore, we developed and validated a rapid LC-MS/MS method to quantify psilocin and its metabolite 4-hydroxyindole-3-acetic acid (4-HIAA) in human plasma. Plasma samples were processed by protein precipitation using methanol. The injected sample was mixed with water in front of a C; 18; analytical column to increase retention of the analytes. Psilocin and 4-HIAA were detected by multiple reaction monitoring (MRM) in positive and negative electrospray ionisation mode, respectively. An inter-assay accuracy of 100-109% and precision of ≤8.7% was recorded over three validation runs. The recovery was near to complete (≥94.7%) and importantly, consistent over different concentration levels and plasma batches (CV%: ≤4.1%). The plasma matrix caused negligible ion suppression and endogenous interferences could be separated from the analytes. Psilocin and 4-HIAA plasma samples could be thawed and re-frozen for three cycles, kept at room temperature for 8 h or 1 month at -20 °C without showing degradation (≤10%). The linear range (R ≥ 0.998) of the method covered plasma concentrations observed in humans following a common therapeutic oral dose of 25 mg psilocybin and was therefore able to assess the pharmacokinetics of psilocin and 4-HIAA. The LC-MS/MS method was convenient and reliable for measuring psilocin and 4-HIAA in plasma and will facilitate the clinical development of psilocybin.