© 2022, Hacettepe University, Faculty of Pharmacy. All rights reserved.Aim: The aim of this experimental study is to investigate the effects of N-ace-tylcysteine and melatonin on cholestasis and their protective effects on liver and renal injury. Materials and Methods: Forty-eight rats were used in the study. Rats were divided into three main groups as sham, main control, and study groups. The main control group is further divided into early sacrification group and control group. The study group is divided into melatonin, N-acetylcysteine, and me-latonin & N-acetylcysteine groups. A laparotomy was performed in study and control groups,and the common bile duct was ligated and divided. Five days after the first operation, blood samples liver and renal tissues were collected from early sacrification group. Between postoperative days five and ten, melatonin, N-acetylcysteine, melatonin & N-acetylcysteine solutions were applied to the rats in the study group subcutaneously and saline to the sham and control group. Blood samples, and liver tissues, and renal tissues of the rest of the rats were collected. Results: AST, ALT, BUN, creatinine, total bilirubin levels were significantly higher in rats with jaundice than in sham group. AST, ALT, total bilirubin, BUN, creatinine levels were significantly higher in the control group at the end of day ten. Among the melatonin group, N-acetylcysteine group, and melatonin & N-acetylcysteine group, all biochemical parameters were not different. Also, the MDA and NO levels were higher in control group in comparison with the study groups. On the other hand, there was no significant difference between the me-latonin group, N-acetylcysteine group, and melatonin & N-acetylcysteine group on behalf of MDA and NO levels, and histopathologic findings. Conclusion: Use of melatonin and N-acetylcysteine in rats with obstructive jaundice prevents damages to free oxygen radicals on the liver and renal tissue.