Background: Tryptophan is an essential amino acid primarily metabolized by the kynurenine pathway in mammals. Intermediate metabolites emerging in this pathway have been associated with many neurogenerative diseases. This study aimed to compare tryptophan pathway metabolite levels in patients with multiple sclerosis (MS) and healthy controls and reveal the relationship of tryptophan metabolites with disease subtype and the Expanded Disability Status Scale (EDSS) score. Methods: The study included a total of 80 MS cases [53 with relapsing remitting MS (RRMS) and 27 with secondary progressive MS (SPMS)] and 41 healthy volunteers. The patients with RRMS were further divided into relapse (RRMS-attack) and non-attack (RRMS-stable) groups. Using liquid chromatography mass spectrometry, tryptophan, kynurenine, kynurenic acid, quinolinic acid, 3-hydroxykynurenine, and 3-hydroxyanthranilic acid levels were measured. The serum metabolite levels of the patient and control groups were compared. In addition, the link and relationship between the EDSS score and disease duration of the patients and their plasma tryptophan metabolite levels were examined. Results: The tryptophan level of the patient group was significantly lower than that of the healthy controls (p<0.05). The kynurenine (105.38±65.43), quinolinic acid (10.42±3.56), kynurenine/tryptophan ratio (0.0218±0.019), and quinolinic acid/kynurenic acid ratio (1.7054±0.96141) of the patients with MS were significantly higher compared to the controls (p<0.05). In the receiver operating characteristic analysis of the power of kynurenine/tryptophan and quinolinic acid/kynurenic acid ratios in predicting the disease, both ratios predicted the diagnosis of MS (area under the curve: 0.793 and 0.645, respectively; p<0.05), albeit at low sensitivity and specificity. The parameters were similar between the RRMS-attack and RRMS-stable patient groups (p>0.05). There was also no significant difference between the RRMS and SPMS patient groups in terms of tryptophan metabolites (p>0.05). Lastly, no significant relationship was observed between tryptophan metabolites and MS subtype and the EDSS score. Conclusion: Our findings revealed that the kynurenine pathway involved in the tryptophan metabolism differed between the patients with MS and healthy controls, and this difference may be a limited guide in the diagnosis of MS, due to major overlaps in values for MS versus Controls, and is insufficient to determine the disease subtype.