Neonatal herpes simplex virus (HSV) can be a devastating illness and may be difficult to diagnose in those cases without a typical skin rash. As a result, physicians often rely on HSV polymerase chain reaction of cerebrospinal fluid to rule out HSV encephalitis. We developed a real-time polymerase chain reaction assay for HSV using the SmartCycler II (Cepheid, Sunnyvale, CA). End point dilution studies showed sensitivity comparable to that of two national reference laboratories that use LightCycler. In-house turnaround time was approximately 1.5 days versus approximately 5.2 days for sending the test to a reference laboratory. We hypothesized that the rapid availability of a negative test result would allow physicians to discharge appropriate patients earlier. Six months after implementation, clinical case analysis identified 12 pediatric patients who were discharged earlier based on more rapid test results, with a projected savings of approximately 55.2 hospital days throughout the first year. Actual length of stay for patients tested in-house was significantly less than that of historical controls and was projected to save approximately 70.2 hospital days in the first year. Including projected annual laboratory cost/test savings of approximately $11,000, a total savings of $38,000 to $43,000 was estimated for the first year of implementation, more than offsetting startup instrument and development cost.