Herpes simplex viruses are significant human pathogens which remain latent in their hosts for life and reactivate to cause disease at or near the initial site of infection. Since HSV infections are associated with high morbidity, rapid detection and diagnosis of these viruses is imperative. While PCR technology for the diagnosis of HSV is now almost utilized universally, virus isolation from mucosa and skin in shell cultures is still applied in many clinical settings. Virus growth in cultured cells alone has the disadvantage that it cannot distinguish between HSV-1 and HSV-2. The aim of this study was to combine these two disparate methods. A protocol was developed to isolate clinical strains directly from the medium used for as the standard source of extraction to isolate DNA. Both HSV-1 and HSV-2 strains were isolated successfully from cervicovaginal swabs transported in OneSwab devices. It was found that the titers of these viruses did not correlate with the C(T) scores determined for the samples using real-time PCR. HSV-2 strains generally had lower titers than those of HSV-1, suggesting that the HSV-2 may be more sensitive to the sample handling procedures. These studies represent the first report of the culture and isolation of HSV from clinical samples using OneSwab intended exclusively for molecular diagnostic analyses.