© The Author(s) 2023. Pacini Editore Medicina and AU-CNS Press.Background. To examine blood S100B and homocysteine levels and to evaluate their possible roles in neurobiological processes in male patients with opioid use disorder. Methods. The study group consisted of 30 male inpatients who were admitted to an outpatient clinic with a diagnosis of “Opioid Use Disorder” according to DSM-5 diagnostic criteria. The control group consisted of 30 healthy male individuals. The first blood samples of the patients were taken at the time of withdrawal symptoms after the psychiatric interview was completed. The patients were started on buprenorphine/ naloxone detoxification therapy. The second blood samples were taken during the stabilization period on the 15th day, with the cessation of withdrawal symptoms. Results. S100B levels were higher in patients during both the withdrawal and stabilization periods compared to those of the control group. Homocysteine levels did not differ between the patient and control groups. S100B and homocysteine levels measured in the patient group did not fluctuate during the withdrawal and stabilization periods. S100B levels increased in both withdrawal and stabilization periods of opioid use disorder while homocysteine values remained unaltered. Conclusions. S100B may play an active role in pathophysiological processes associated with opioid use disorder. S100B levels remained high during the treatment, suggesting that the compensatory process continued throughout its duration.