BACKGROUND: Acute appendicitis is the most common abdominal surgical emergency in children, and appendectomy is the most frequent acute abdominal operation. Prompt diagnosis and surgical treatment are required to reduce the risk of perforation and prevent complications, especially in small children. Enterochromaffin cells that contain large amounts of serotonin are mostly located in the distal appendix. Serotonin metabolite 5-hydroxyindoleacetic acid (5-HIAA) could therefore be a marker for acute appendicitis. OBJECTIVE: We tested urinary 5-HIAA concentrations in spot urine samples from children with acute appendicitis. METHODS: We enrolled 93 patients who underwent surgery for suspicion of acute appendicitis. The diagnosis was made intraoperatively and confirmed histopathologically. Additionally, urine samples from 102 healthy children were collected as controls. Their 5-HIAA was measured using high-performance liquid chromatography. RESULTS: Acute appendicitis was diagnosed in 81 patients, whereas there were other explanations for abdominal pain in the remaining 12 patients in the non-appendicitis group. The control group comprised 102 healthy children. Considering the median of all measured 5-HIAA values as the cut-off, we analysed the proportions of patients with elevated values in all the groups. Our analysis showed that statistically there was no significant difference in the distribution of percentages among the groups. The area under the curve for 5-HIAA was 0.55 (95% CI 0.47 to 0.62) with sensitivity and specificity 60.4% and 48.9%, respectively. CONCLUSIONS: Urine 5-HIAA concentration measured in spot samples is not a reliable method for diagnosing acute appendicitis in children.