Background: Familial Mediterranean fever (FMF) is an autoinflammatory syndrome characterized by recurrent episodes of fever and aseptic polyserositis. Subclinical inflammation generates a hidden threat to the development of FMF complications such as amyloidosis in attack-free intervals. The kynurenine pathway (KP) has been considered an important player in inflammation and immune response. The study was aimed to measure serum levels of KP metabolites in patients with FMF in the attack-free period. Methods: A total of 161 participants were recruited from the rheumatology department in this single-centre, case–control study. Participants meeting the eligibility criteria were divided into healthy controls (n = 80) and FMF (n = 81). The laboratory data were obtained from the electronic registration database. Serum tryptophan (TRP), kynurenine (KYN), kynurenic acid (KYNA), 3-hydroxyanthranilic acid, 3-hydroxykynurenine (3HK), and quinolinic acid (QUIN) concentrations were measured with tandem mass spectrometry. Laboratory findings of FMF patients and healthy controls subjects were compared and evaluated. Results: Serum TRP and KYNA levels were significantly decreased in both FMF groups compared to the control group, while the levels of KYN, QUIN, 3HK, the KYN/TRP ratio, and red cell distribution width were higher. Conclusion: TRP degradation by the KP is increased in patients with FMF. KP metabolites can be useful in demonstrating subclinical inflammation.