Abstract: OBJECTIVE To establish a method for simultaneous determination of valproic acid, phenobarbital and topiramate in plasma of epileptic children by high performance liquid chromatography-tandem mass spectrometry(HPLC-ESI-MS/MS). METHODS The samples were pre-treated by precipitation protein method with organic solvent. Valproic acid-D6, topiramate-D12, and phenobarbital-D5 were used as internal standard. Chromatographic column: Phenomenex Kinetex^® EVO C₁₈(2.1 mm×50 mm, 2.6 μm); flow phase: 0.07% formic acid-1 mmol ammonium acetate-water(A) and methanol(B), gradient elution; flow speed: 0.5 mL·min^-1. Ion source was electric spray ion source, multireaction monitoring mode for quantitative analysis, using negative ion monitoring mode. The specificity, lower limit of quantitation(LLOQ), standard curve, residual effect, dilution effect, accuracy, precision, matrix effects and stability were investigated. RESULTS The linear range of valproic acid was 0.6-120 μg·mL^-1 (r=0.998 9), and LLOQ was 0.6 μg·mL^-1. The linear range of phenobarbital was 0.25-50 μg·mL^-1 (r=0.999 1), and LLOQ was 0.25 μg·mL^-1. The linear range of topiramate was 0.25-50 μg·mL^-1 (r= 0.999 0), and LLOQ was 0.25 μg·mL^-1. The intra-batch and inter-batch precision RSD was ≤8.0%, with good stability, and this method was not affected by common matrix and hemolysis matrix(hemolysis degree ≤5%). The methodology validation was in line with the provisions of Chinese Pharmacopoeia(2020 edition). CONCLUSION This study determined the blood concentration of valproic acid, phenobarbital and topiramate in children with HPLC-ESI-MS/MS, which is rapid, simple, stable, economical, and high sensitivity and specificity. It can be applied to therapeutic drug monitoring of valproic acid, phenobarbital and topiramate in clinical children with epilepsy.