BACKGROUND: Gentamicin is a commonly used medication in NICUs. It is known to have ototoxic & nephrotoxic side effects. To date there is no consensus about dosing regimen in different institutions. Our study aims to evaluate the Neofax® dosing regimen for gentamicin in neonatal early onset sepsis in relation to trough level before the second dose and its association with the incidence of gentamicin side effects, namely hearing impairment/loss and acute kidney injury. METHODS: Retrospective chart review of newborns admitted to Tawam hospital NICU (June 2019–May 2020) who received gentamicin for early onset sepsis (≤72 hours old). Trough levels before the second dose at 24,36 and 48 hours were reviewed (≥1 mg/L is high). Excluded patients with renal risk factors. Side effects (hearing impairment, acute renal injury) were also assessed. RESULTS: Total of 265 infants were included, among whom 149 patients received gentamicin at 24 hours interval, 99 at 36 and 17 at 48 hours interval. Trough level was high in 76% (P = 0.022), 65% (P = 0.127), and 53% (P = 0.108) of patients who received gentamicin at 24, 36, and 48 hours, respectively. Hearing screening was normal in 99.2% of patients, while 2 patients failed the test (Both with normal trough levels). No patients in our study developed renal injury related to gentamicin use. CONCLUSION: Neofax® gentamicin dosing often results in high trough levels, especially in late preterm/term infants. This study found no correlation between high trough levels and hearing impairment upon discharge or acute kidney injury. Further studies with larger sample size are recommended.