Introduction: Gentamicin is the first line antibiotic for early onset neonatal sepsis. Yet the problem with gentamicin is the low therapeutic index. It is known that high trough levels correlate with ototoxicity and nephrotoxicity while the peak levels decide the therapeutic effect. Once daily dose (ODD) and twice daily dose (TDD) schedules are practised in neonatal units in Sri Lanka. Objectives:To assess whether the ODD and TDD schedules of gentamicin used in neonates are effective in avoiding high serum trough levels and in achieving peak serum levels in the therapeutic range.Method: A prospective study was conducted from April to August 2017 in four neonatal units on term neonates less than one week old, in whom gentamicin was started (TDD regime= 2.5 mg/kg 12 hourly and ODD regime= 5mg /kg 24 hourly) during the study period. Trough and peak serum gentamicin levels (SGLs) were assessed before and after the third dose in the TDD regime and after the second dose in the ODD regime. Serum creatinine (SCr) was measured in all the neonates and neonates with high trough levels were referred for hearing assessment.Results: A total of 50 neonates of clinical maturity 37-41 weeks and birth weight 1.8-4.1 kg was included. High trough levels (>2 mg/dl) were seen in 48% of the TDD group and in 12% of the ODD group (p=0.005) Mean trough value in the ODD group was 1.15 mg/dl (SD 0.87) and in the TDD group was 2.14 mg/dl (SD 1.72). (T=-2.564, df=35, p = 0.015). Peak SGL in the therapeutic range (5-_________________________________________ The authors declare that there are no conflicts of interest Funding: University research grant for financial assistance. Open Access Article published under the Creative Commons Attribution CC-BY License 10 mg/dl) was seen in 92% of ODD regime and in 80% of the TDD regime. Mean peak values in the ODD and TDD regimes were 9.42 (SD 2.37) mg/dl and 7.12 (SD 2.18) mg/dl respectively (T= 3.56, df=48, p = 0.001). SCr and the hearing assessment were within normal limits in all neonates. Conclusions:The ODD gentamicin schedule of 5 mg/kg achieves a significantly higher peak serum gentamicin level and safer trough levels than the TDD schedule of 2.5 mg/kg. DOI: http://dx.doi.org/10.4038/sljch.v48i1.8647 (