Background Selective serotonin reuptake inhibitors (SSRIs) block the serotonin transporter on neurons, but also on platelets, thus decreasing platelet serotonin concentrations in users of SSRIs. Data on plasma-free serotonin concentrations in SSRI users are lacking, while plasma-free serotonin is available for receptor binding and plays a role in several pathophysiological processes. We therefore measured the plasma-free and platelet serotonin concentrations in users of SSRIs and age-matched healthy controls, and we analysed plasma concentrations of the serotonin precursor tryptophan and serotonin metabolite 5-hydroxyindoleamineacetic acid (5-HIAA). Methods For this cross-sectional single-centre case control study, participants were recruited at the departments of Psychiatry and General Medicine. High-performance liquid chromatography combined with tandem mass spectrometry (LC-MS/MS) was used to measure plasma-free and platelet serotonin, plasma tryptophan and 5-HIAA concentrations. Preanalytical conditions were optimized by careful blood collection, rapid sample handling, high-speed centrifugation, drug and diet restrictions and age-matched controls. Results In 64 SSRI users, median concentrations of plasma-free and platelet serotonin were 10-fold and 14-fold lower, respectively, than in 64 matched controls. Patients using higher dose SSRIs or those with higher affinity for the serotonin transporter had lower plasma-free and platelet serotonin concentrations. Compared with controls, SSRI users had similar median plasma tryptophan concentrations but slightly higher plasma 5-HIAA concentrations. Conclusion SSRI users have low platelet serotonin and low plasma-free serotonin. This could not be explained by lower concentrations of its precursor tryptophan, and only partially by increased breakdown to 5-HIAA.