Tryptophan metabolism plays a key role in several (patho)physiological conditions. In order to study the clinical importance of tryptophan and its predominant metabolites (kynurenines), it is important to be able to measure large series of samples with high accuracy and reliability. We aimed to develop a high-throughput on-line solid-phase extraction-liquid chromatographic-tandem mass spectrometric (XLC-MS/MS) method that enables the measurement of tryptophan and its metabolites kynurenine and 3-hydroxykynurenine in plasma. Fifty microliters plasma equivalent was pre-purified by automated on-line solid-phase extraction, using strong cation exchange (PRS, propylsulphonic) cartridges. Chromatographic separation of the analytes and deuterated analogues occurred by C18 reversed phase chromatography. Mass spectrometric detection was performed in the multiple reaction-monitoring mode using a quadrupole tandem mass spectrometer with positive electrospray ionization. Total run-time including sample clean-up was 8 min. Intra- and inter-assay analytical variations were less than 9%. Linearity in the 0.11-1200 (tryptophan) and 0.050 and 0.023-45 micromol/L (kynurenine and 3-hydroxykynurenine, respectively) calibration range was excellent (R>0.99). Detection limits were 30 nmol/L for tryptophan, 1 nmol/L for kynurenine and 5 nmol/L for 3-hydroxykynurenine. Reference intervals for 120 healthy adults were 45.5-83.1 micromol/L (tryptophan), 1.14-3.02 micromol/L (kynurenine), <0.13 micromol/L (3-hydroxykynurenine) and 19.0-49.8 for tryptophan-to-kynurenine ratio. Blood sampling for tryptophan and tryptophan-to-kynurenine ratio should be performed before breakfast, due to biological variation during the day. This study describes how plasma tryptophan, kynurenine and 3-hydroxykynurenine can be measured accurately and precisely by automated high-throughput XLC-MS/MS.