The use of empiric acyclovir for suspected neonatal herpes simplex virus (HSV) infection has been debated for years. To identify the gap in the decision to initiate empiric acyclovir, we performed a retrospective chart review and administered a survey to pediatricians to assess current practices regarding evaluation for possible HSV infection. Seventy infants received empiric acyclovir over a 1-year period; of these, 3 infants (4.3%) had positive HSV testing. Fourteen infants were identified as “high-risk” for HSV infection; of these, 13 infants had incomplete testing. Survey results revealed uncertainty in the decision to initiate acyclovir and in the composition of complete diagnostic testing. This study confirmed the clinical uncertainty in the decision to initiate empiric acyclovir. Using this chart review and survey as a baseline, future efforts will focus on a quality improvement project to reduce empiric acyclovir use in low-risk infants and to ensure complete diagnostic evaluation in high-risk infants.