Aim: This study was conducted to optimize gentamicin (genta) use in paediatrics incorporating the estimation of serum drug concentrations and customized pharmacokinetic (PK) simulations. Method: 66 patients (age 1-144 month) were enrolled in the study. They received gentamicin 2-2.5 mg/kg every 8 hr. (TID regimen). Serum genta concentrations within one dosing interval of dose 3 and dose 4 were determined using an immunoassay; blood samples were collected at 30 minutes after the end of infusion while a trough level sample was collected just before the next dose. Customized PK simulation analysis was based on the following assumptions: Single-compartment model, first-order elimination, and repeated short time IV infusion. PK parameters and simulation of genta peak/trough levels after various regimens were estimated and compared statistically. Results: About 65% of the patients showed subtherapeutic peak genta levels (less than 6 ug/ml) during the dosing intervals. Potentially toxic trough levels (>1 ug/ml) were observed in two patients. Neonates (1-12 months) showed a relatively higher mean genta volume of distribution (Vd), 0.51±0.18 L/Kg, vs 0.37±0.13 (p < 0.05) in children (>1-12 y). Half-life in both groups was comparable (about 3 h). Simulation suggests BID regimens will provide the best theoretically overall peak/trough targets. Discussion: In children, a higher volume of distribution of genta could be associated with the lower serum peak levels due to the conventional dosing regimen (TID). Conclusion: Optimal dosing regimen in pediatric patients can be designed to achieve target high peak, low trough levels based on simplified customized PK simulations.