Abstract Background: Sepsis remains one of the leading causes of neonatal morbidity and mortality, particularly among premature infants. Blood culture is the ‘gold standard’ for the diagnosis of neonatal sepsis but is associated with several pitfalls. Aim of the work: We aim to evaluate the validity of measuring serum (S.Hep) and urinary hepcidin (U.Hep) concentrations as diagnostic biomarkers for late-onset sepsis (LOS) in preterm infants. Patients and Methods: The current case-control study included 73 cases of clinically and laboratory confirmed late-onset sepsis as the ‘case group’ and 50 nonseptic premature infants of comparable age and sex as the ‘control group’. S.Hep and U.Hep concentrations were evaluated at enrollment (acute sample) and after 1 week of treatment (convalescent sample). Results: Patients had a statistically significant higher concentration of both S.Hep and U.Hep as compared with nonseptic controls (p = 0.0001). Similarly, a significant reduction of both S.Hep and U.Hep was detected after 1 week of treatment (p=0.001). A cut-off value of⩾94.8 ng/ml of S.Hep and⩾264 ng/mg of U.Hep/urinary creatinine showed high sensitivity, specificity, and positive predictive value in the diagnosis of neonatal LOS. Conclusions: Both S.Hep and U.Hep can function as promising accurate and rapid surrogate tests for the diagnosis of LOS. U.Hep measurement has the advantage of being noninvasive, with no hazards of phlebotomy, and is less variable throughout the day.