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Early-onset sepsis is a common diagnosis in neonatal intensive care units. Because of the low incidence, overtreatment is also common.To measure the sensitivity and negative predictive value of 2 serial white blood cell counts and a negative blood culture at 24 hours in predicting a noninfected neonate in the evaluation of early-onset sepsis.We performed a historical cohort study of neonates in the University of Massachusetts Newborn Nursery and neonatal intensive care unit born between 1999 and 2008 who had sepsis evaluations within the first 24 hours of life.Three thousand two hundred thirteen patients were identified; 59 were excluded due to missing data. Of the 3154 included neonates, 1539 (49%) had 2 normal immature to total neutrophil (I:T) ratios and a negative blood culture at 24 hours. Two of these blood cultures showed growth of bacteria after 24 hours but were considered contaminants, and antibiotics were stopped at 48 hours. None of the 1539 neonates with normal I:T ratios was subsequently diagnosed with sepsis (negative predictive value 100%; [95% confidence interval: 99.905%–100%]).In this study, the combination of 2 serial normal I:T ratios and a negative blood culture at 24 hours in the evaluation of early-onset sepsis shortly after birth is indicative of a noninfected neonate. This suggests that antibiotics can safely be stopped at 24 hours in these neonates, which comprises approximately 50% of our study population.