The aim of the present study was to characterize the pharmacokinetic profile of oral ibuprofen on consecutive 3 d by trough serum levels, and if possible to define a cut-off level for ductal closure in preterm infants. The study enrolled 20 preterm infants with gestational age ≤30 weeks, birth weight <1250 g and hemodynamically significant patent ductus arteriosus (hsPDA). Patients received oral ibuprofen at an initial dose of 10 mg/kg, followed by 5 mg/kg at 24 and 48 h. Patients were compared for serum ibuprofen levels in addition to their demographic and clinical data in case of their response to the treatment. hsPDA closed in 16 (80%) of the patients. Although mean ibuprofen levels on consecutive 3 d showed a plateau in general, ibuprofen serum levels on the first treatment day were statistically low in patients with unclosed hsPDA (p = 0.003). The optimal cut-off value for serum ibuprofen level on the first treatment day was measured as 5.5 mg/l with 100% sensitivity and 93% specificity. Serum ibuprofen level on the first treatment day seems to be an important factor for a successful ductal closure. Target concentration approach by the evaluation of trough level may be applicable to real-time dosing strategy.