Background: The aim of this study was to investigate the protective potency of melatonin, amifostine (WR-2721), and N-acetylcysteine (NAC) when administered intraperitoneally (i.p.) 15 min before 10-Gy single-fraction radiotherapy. Materials and Methods: In this study, 35 female Sprague Dawley rats were divided into five groups of seven rats each. The rats in the control group did not receive any treatments. Rats in the radiotherapy, melatonin, amifostine, and NAC groups underwent abdomino-pelvic irradiation with 10-Gy single fraction gamma (γ) irradiation. Melatonin 50 mg/kg, amifostine 200 mg/kg, and NAC 500 mg/kg were i.p. administered to the rats 15 min before irradiation. Animals were sacrificed 48 h after irradiation. Uterus samples were collected and, routine histopathological tissue processing was performed. Sections from tissue samples were stained with H&E and analyzed with the terminal deoxynucleotidyl transferase dUTP nick end labelling method (TUNEL assay). Results: Severe morphological degenerations and increases in the apoptotic index (AI) were observed in the radiotherapy group. Tissue protection and AI reduction were observed in the amifostine and NAC groups. Melatonin was more effective than amifostine and NAC. Morphological damage was almost completely repaired, and the AI of the melatonin group was quite similar to that of the control group. Conclusion: This experiment failed to determine a more successful administration technique of amifostine. The protective effects of amifostine and NAC were similar. Melatonin was more successful than these two drugs, and might be an alternative to amifostine when time, dose, or adverse effect constraints are encountered.