Background The European Society for Blood and Marrow Transplantation (EBMT) recommends a busulfan target cumulative area under the curve (cAUC) of 78-101 mg·h/L for hematopoietic stem cell transplantation (HSCT). Currently, no population pharmacokinetic (PopPK)-optimized four-times-daily (Q6H) regimen reliably achieves this range. We developed a PopPK-guided dosing protocol to address this gap. Methods Clinical and demographic data from pediatric HSCT recipients receiving busulfan were retrospectively analyzed to develop a PopPK model. Bayesian estimation identified the optimal busulfan dose (0.50-1.25 mg/kg/dose) for target attainment in pediatric patients (age 0.5-18 years; weight 5-80 kg). Finally, the proposed dosing strategy was validated using an independent retrospective cohort. Results The PopPK model was developed using data from 65 pediatric patients. Age and body weight identified as significant covariates. Optimized dosing recommendations for the Q6H × 4 days busulfan regimen were established through Bayesian estimation. For validation, two cohorts were retrospectively analyzed: (1) weight-based dosing (n=19) and (2) model-informed dosing (n=15). The groups demonstrated comparable distributions in both age ( p=0.78) and weight ( p=0.63). Notably, the model-informed group achieved significantly higher cAUC values (mean difference, 18.0 mg·h/L; 95% confidence interval , 9.83-26.1 mg·h/L; p < .001), with 67% of these patients reaching the target exposure range. Conclusion We developed a Q6H × 4 days busulfan dosing regimen through PopPK modeling. For a representative 6-year-old patient with a body weight of 20 kg, the recommended dose is 1.25 mg/kg/dose. Therapeutic drug monitoring following the initial dose remains clinically essential.