Background <p>Busulfan is a chemotherapeutic agent used in conditioning regimens before hematopoietic stem cell transplantation (HSCT) in pediatric patients. Due to its pharmacokinetic variability, therapeutic drug monitoring (TDM) is indispensable to ensure favorable outcomes and minimize risks.</p> Methods <p>A retrospective observational study was conducted on 166 pediatric patients receiving busulfan at King Abdullah Specialist Children’s Hospital from July 2020 to May 2024. Busulfan concentration was determined using chromatographic separation with Waters Acquity UPLC^® followed by detection on a Xevo TQ-MS tandem quadrupole LC-MS/MS. The analysis included demographic data, busulfan dosing, therapeutic levels, and clinical outcomes such as length of hospital stay and mortality. Factors influencing busulfan levels were also assessed.</p> Results <p>Among the 166 patients analyzed, 41.57% were aged 6–11 years, and 59.64% were male. Sickle cell anemia was the predominant indication for busulfan use (72.28%). The therapeutic level was achieved in 42.77% of patients, while 42.17% were below the range. Gender significantly influenced AUC values (males: 898.01 µmol·min/L; females: 1000.10 µmol·min/L, <i>p</i> = 0.0091). Mortality was observed in 4.82% of patients.</p> Conclusion <p>The study highlights the variability in busulfan pharmacokinetics and its clinical implications in pediatric patients. Gender differences and subtherapeutic exposure were associated, emphasizing the need for individualized dosing and rigorous monitoring to optimize therapy. Among 166 patients, 42.77% achieved therapeutic levels, 42.17% were subtherapeutic, and mortality was 4.82%. A significant gender difference was observed, with females exhibiting higher AUC values than males (<i>p</i> = 0.0091).</p> Clinical trial number <p>Not applicable.</p>