Objectives: Recent studies have shown that derangements in kynurenine pathway metabolite levels are associated with various pathologies such as neurodegenerative diseases, schizophrenia, depression, bipolar disorder, rheumatoid arthritis, and cancer. Therefore, reliable, accurate, fast, and multiplex measurement methods for kynurenines have become increasingly important. This study aimed to validate a new mass spectrometric method for analyzing tryptophan metabolites. Methods: A tandem mass spectrometric method, including protein precipitation and evaporation steps, was developed to measure serum levels of tryptophan, kynurenine, kynurenic acid, 3-hydroxykynurenine, and 3-hydroxyanthranilic acid. Samples were separated using a Phenomenex Luna C18 reversed-phase column. The kynurenine pathway metabolites were detected by tandem mass spectrometry. The developed method was validated according to Clinical & Laboratory Standards Institute (CLSI) guidelines and applied to hemodialysis samples. Results: The developed method was linear at the concentrations of 48.8 – 25,000, 0.98 – 500, 1.2–5000, 1.2–5000, and 0.98–250 ng/mL for tryptophan, kynurenic acid, kynurenine, 3-hydroxyanthranilic acid, and 3-hydroxykynurenine, respectively. The imprecisions were less than 12 %. The median serum concentrations of tryptophan, kynurenine, kynurenic acid, 3-hydroxykynurenine, and 3-hydroxyanthranilic acid were 10530, 1100, 218, 17.6, and 25.4 ng/mL in pre-dialysis blood samples, respectively. They were 4560, 664, 135, 7.4, and 12.8 ng/mL in post-dialysis blood samples, respectively. Conclusions: A fast, simple, cost-effective, accurate, robust, and validated tandem mass spectrometric method was developed, and the method was successfully used for the quantitation of kynurenine pathway metabolite concentrations in hemodialysis patients.