Background Serotonin is used for the diagnosis and follow-up of neuroendocrine tumors (NET). We describe the analytical and clinical validation of a liquid chromatography tandem mass spectrometry (LC-MS/MS) based serotonin assay for serum and platelet-rich plasma (PRP). Methods An LC-MS/MS based method for serum and PRP serotonin was validated by determination of assay imprecision, carry-over, linearity, interference, recovery, sample stability and a matrix/method comparison of serum and PRP serotonin was made with whole blood serotonin. Furthermore, upper limits of normal were determined and serotonin concentrations of healthy individuals, 14 NET patients without evidence of disease and 51 NET patients with evidence of disease were compared. Results For serum and PRP fractions, total assay imprecision was <5%. All correlation coefficients were 0.98 and the serum and platelet-rich serotonin upper limit of normal were 5.5nmol/109 platelet and 5.1nmol/109 platelet, respectively. NET patients with confirmed evidence of disease had significantly higher serum and PRP serotonin levels when compared to NET patients without evidence of disease and healthy volunteers. Conclusions LC-MS/MS based serum and PRP serotonin assays were developed with suitable analytical characteristics. Furthermore, serum and PRP serotonin was found to be useful for monitoring NET patients.