Background: Pediatric patients undergoing diagnostic tests in the pediatric emergency room are frequently sedated. Although efforts are made to prevent adverse events, no sedation protocol has specified the optimal regimen, dosage, and interval of medication to prevent adverse events. This study analyzed the safety and efficacy of sequential pediatric sedation protocols for pediatric patients undergoing diagnostic tests in the pediatric emergency room of a single tertiary medical center.Methods: The medical records of patients aged <18 years who visited the pediatric emergency room of Seoul Asan Medical Center between January and December 2019 for diagnostic testing were retrospectively reviewed. Sedation protocols consisted of 50 and 25 mg/kg chloral hydrate, 0.1 and 0.1 mg/kg midazolam, and 1 and 0.5–1 mg/kg ketamine, administered sequentially at intervals of 30, 20, 10, 10, and 10 min, respectively. Patients were assessed prior to sedation, and adverse events were investigated.Results: Of the 289 included patients, 20 (6.9%) experienced adverse events, none serious, and nine (3.1%) failed to reach the depth of sedation required to complete the test. The regimen (P=0.622) and dosage (P=0.777) of the sedatives were unrelated to the occurrence of adverse events when sedation was performed according to protocol.Conclusion: The sedation protocol used in these patients, consisting of sequential administration of minimum dosages, achieved a sufficient depth of sedation with relatively few adverse events, indicating that this protocol can be used safely and effectively for painless sedation in pediatric patients undergoing diagnostic testing.