Objectives: This study aims to investigate the effect of smoking on the metabolism of kynurenine and thus contribute to the elucidation of the potential mechanism of cigarette smoking. Methods: The study included 82 smokers and 63 nonsmokers who applied to the Family Medicine Polyclinic for routine check-ups. Sociodemographic data, routine laboratory results, Framingham risk scores (FRS), and Fagerström Nicotine Dependence Test (FTND) were recorded. Serum tryptophan, kynurenine, kynurenic acid, 3-hydroxyanthranilic acid, 3-hydroxykynurenine, and quinolinic acid concentrations were measured with tandem mass spectrometry. Results: Serum tryptophan levels (p=0.040) were statistically significantly lower in smokers, and the kynurenine/ tryptophan ratio and serum kynurenine, kynurenic acid, quinolinic acid, 3-hydroxyanthranilic acid levels were higher (p<0.001). The correlation analysis in the smoker group showed a positive correlation between serum kynurenic acid levels and FTDN. The serum kynurenine levels were positively correlated with the levels of total cholesterol, triglyceride, low-density lipoprotein cholesterol, and FRS. There was a positive correlation between serum quinolinic acid levels and participants’ systolic and diastolic blood pressures. Conclusions: Our findings showed that tryptophan metabolism via the kynurenine pathway was induced in smokers.