Purpose Acute appendicitis is one of the most common surgical emergencies. Diagnosis is usually made depending on the presenting history, clinical evaluation, and laboratory tests. The aim of this study was to investigate the role of urinary 5-hydroxyindoleacetic acid (U-5-HIAA) in the early diagnosis of acute appendicitis. Methods Thirty-five pigmented male rabbits were divided into 5 groups. Group 1 is the control (n = 7); group 2 is the sham (n = 10). The appendix was ligated from its base, and an appendectomy was performed after 12, 24, 36 hours in group 3 (n = 7), group 4 (n = 7), and group 5 (n = 7), respectively. Spot urine samples were obtained for U-5-HIAA determination, and appendectomy tissues were examined histopathologically. Results Acute appendicitis was diagnosed in all animals in group 3, group 4, and group 5, and the mean levels of U-5-HIAA in group 3 were higher than in the other groups. The mean of U-5-HIAA levels between animals with appendicitis and those without showed a significant difference ( P = .003). The U-5-HIAA cutoff point of 4.15 mg/g creatinine had a sensitivity of 85%, a specificity of 64.29%, and an accuracy of 76% (area under curve = 0.805) for acute appendicitis. The probability of acute appendicitis is found to be 10, 2 times more when the U-5-HIAA level is greater than 4.15 mg/g creatinine. Conclusion We have concluded that spot U-5-HIAA level increases significantly in the early stages of acute appendicitis.