Amikacin is widely used in the treatment of suspected or confirmed neonatal infections. However, dosage regimens are not well defined in this group of patients because of a wide inter-individual pharmacokinetic variability. An individualized goal-oriented amikacin dosage design was applied using population pharmacokinetic data. A dosing chart was developed for neonates during the first 2 days of life, by using population pharmacokinetic parameter values and USCPACK software. This dosing chart based on gestational age (GA) and body weight gives a once-a-day amikacin dosage regimen involving an injection every 24 h. Validation was performed in 57 neonates less than 2 days old, divided into three GA groups and prospectively treated using the dosing chart. Target peak serum levels of amikacin were obtained in 62-80% of patients after the first dose and in 80-100% after the second dose, and trough concentrations were obtained in 100%. This study has confirmed the need for individualization of amikacin dosage regimens in neonates.