Aim: The purpose of this study was to assess the effect of administering Nerium oleander distillate on hypercholesterolemic rats' serum biochemical parameters. Materials and Methods: Three equal groups of 30 male Sprague-Dawley rats were formed. No application was made for the control group. The second group received a high cholesterol diet, while the third group received a high cholesterol diet in addition to lyophilized Nerium oleander distillate by oral gavage once daily. After three months, blood was drawn after sedation and they were euthanized. Serum fasting blood glucose, triglyceride, high-density lipoprotein, total cholesterol, low-density lipoprotein, alanine aminotransferase, aspartate aminotransferase, albumin, and total protein levels, as well as alkaline phosphatase, lactate dehydrogenase, creatinine, and urea levels, were determined using an autoanalyzer. Results: Alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, cholesterol, glucose, low-density lipoprotein, and triglyceride levels increased (p < 0.05) in the group fed a high cholesterol diet compared to the control group, but Nerium oleander treatment decreased the elevated aspartate aminotransferase, cholesterol, low-density lipoprotein, and triglyceride (p < 0.05). Conclusion: To summarize, it can be stated that Nerium oleander distillate has antihypercholesterolemic and liver-protective properties.