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Background and Objectives Despite the growing popularity of medicinal plants, concerns remain regarding their potential side effects and toxicity, particularly on organs like the liver and kidneys. This study aims to evaluate the toxic effects of Citrulus colocynthis on liver enzymes and kidney biochemical markers in rats. Subjects and Methods In this study, 40 adult male rats weighing between 250-300 grams were randomly assigned to four groups: a control group and three experimental groups receiving Citrulus colocynthis extract at doses of 100 mg/kg, 200 mg/kg, and 400 mg/kg, respectively. Citrulus colocynthis extract was given through gavage, and after the completion of the test, the serum levels of liver enzymes and several renal factors were measured. Results The results of this study indicated that all three doses of the extract caused a significant increase in alkaline phosphatase and gamma-glutamyl transferase levels compared to the control group (p < 0.05). Additionally, different doses of the extract led to an increase in serum levels of urea, creatinine, and uric acid; however, this increase was not statistically significant compared to the control group (p > 0.05). Conclusion Although the extract of medicinal plants such as Citrulus colocynthis helps in the treatment of diabetes, asthma, allergies, cancer and colds, its toxicity on organs such as liver and kidney, which are vital organs of the body, should be studied more comprehensively. [ABSTRACT FROM AUTHOR]