Neonatal Sepsis (NS), sepsis occurring in children up to one month of life, is a leading cause of neonatal deaths in developing countries. The incidence of neonatal sepsis is higher in premature and low-birth weight infants, although most cases occur in full-term infants. Furthermore, the empiric treatment of patients with bloodstream infections (BSI) has become more complicated in an era of increasing antimicrobial resistance. Hence, the aim of this review article is to show the problem arising while using ampicillin plus gentamicin as mainstay therapy. A number of risk factors has been mentioned to increase the incidence of NS, in Early onset neonatal sepsis (EONS) maternal and neonatal factors (maternal infections, nutritional status of the mother, premature deliver, early rupture of membrane, prolonged delivery, underweight) has been implicated while in late onset neonatal sepsis (LONS) environmental factor (instrumental delivery, hospital admission…) taking the lead to cause NS. One study analyzed 19 different studies from 13 different developing countries on prevalence of microbes for causing sepsis in neonates and reported that, Staphylococcus aureus, Klebsiella spp. and Escherichia coli accounted for 55% (39-70%) of culture positive sepsis. Empirical treatment for neonatal sepsis, recommended in the current WHO guidelines is intravenous ampicillin (or penicillin) plus gentamicin for 7 days. The clinical questions are how reliable to use ampicillin plus gentamicin in all settings? Are there any reports contrary to these guidelines? What should be the dosing interval of Gentamicin/ampicillin? Some clinicians argue that, since majority of mothers in current practice are giving births at hospital setups, where Staphylococcus aureus is predominating, treatments of the standard guideline for NS has to be modified. Therefore, it will be important to evaluate all newly developed antibiotics in neonates to assure their maximum efficacy and safety.