Subclinical hypothyroidism (SCH) is a usually asymptomatic endocrine disorder characterized by elevated thyroid-stimulating hormone (TSH) and normal free thyroxine (fT4) levels. The kynurenine pathway is activated during inflammation, and several of its metabolites have been proposed as potential biomarkers in inflammatory diseases. This study aims to understand the pathophysiology of the disease by investigating changes in the metabolism of kynurenine in patients with SCH and evaluating the potential of these metabolites as biomarkers. The study included 44 patients diagnosed with SCH in the endocrinology clinic and 35 healthy volunteers. Liquid chromatography-tandem mass spectrometry (LC–MS/MS) was used to analyze the metabolites of the kynurenine pathway. TSH, anti-thyroid peroxidase (anti-TPO) and anti-thyroglobulin (anti-TG) antibody levels were significantly higher in the SCH group than in the control group (p < 0.05). Tryptophan (TRP) levels were significantly lower in the SCH group, while kynurenic acid (KYNA), 3-hydroxykynurenine (3-HKYN) and 3-hydroxyanthranilic acid (3-HAA) levels were significantly higher (p < 0.05). In the ROC analysis, the KYNA/TRP ratio (AUC: 0.827, 95% CI 0.737–0.917) had the highest discriminatory power to distinguish individuals with SCH, followed by 3-HAA/TRP (AUC: 0.758, 95% CI 0.655–0.862), KYNA/kynurenine (KYN) (AUC: 0.718, 95% CI 0.604–0.833) and TRP (AUC: 0.713, 95% CI 0.594–0.833). 3-HKYN showed the highest value with a sensitivity of 86%, while the KYNA/TRP ratio stood out with a specificity of 97%. The KYNA/TRP ratio may serve as a potential biomarker candidate for SCH. The interaction of kynurenine metabolism with inflammatory processes may play a role in the development of SCH. These findings suggest that alterations in the kynurenine pathway may contribute to the pathophysiology of SCH and help to better understand the underlying mechanisms of the disease.