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Even in the era of effective antiviral therapy, neonatal herpes simplex viral infection causes significant morbidity and mortality in newborns. Prompt diagnosis is the cornerstone of treatment of these infants. Outside and inside the neonatal clinical practice, polymerase chain reaction (PCR) is replacing culture as a method of facilitating a speedy diagnosis of herpes simplex virus infection. New pediatric guidelines call for testing of high-risk asymptomatic infants, and thus, many more surface cultures and PCRs are being performed. This review aims to comprehensively describe the perinatal transmission of herpes simplex virus infection and the clinical presentation of neonatal herpes simplex disease, as well as to discuss whether PCR remains superior to culture methods.