ObjectiveAcute infection is the most common presentation of children in primary care with only few having a serious infection (eg, sepsis, meningitis, pneumonia). To avoid complications or death, early recognition and adequate referral are essential. Clinical prediction rules have the potential to improve diagnostic decision-making for rare but serious conditions. In this study, we aimed to validate a recently developed decision tree in a new but similar population.DesignDiagnostic accuracy study validating a clinical prediction rule.Setting and participantsAcutely ill children presenting to ambulatory care in Flanders, Belgium, consisting of general practice and paediatric assessment in outpatient clinics or the emergency department.InterventionPhysicians were asked to score the decision tree in every child.Primary outcome measuresThe outcome of interest was hospital admission for at least 24 h with a serious infection within 5 days after initial presentation. We report the diagnostic accuracy of the decision tree in sensitivity, specificity, likelihood ratios and predictive values.ResultsIn total, 8962 acute illness episodes were included, of which 283 lead to admission to hospital with a serious infection. Sensitivity of the decision tree was 100% (95% CI 71.5% to 100%) at a specificity of 83.6% (95% CI 82.3% to 84.9%) in the general practitioner setting with 17% of children testing positive. In the paediatric outpatient and emergency department setting, sensitivities were below 92%, with specificities below 44.8%.ConclusionsIn an independent validation cohort, this clinical prediction rule has shown to be extremely sensitive to identify children at risk of hospital admission for a serious infection in general practice, making it suitable for ruling out.Trial registration numberNCT02024282.