Rapid confirmation of herpes simplex virus (HSV) is essential in many clinical settings. Viral isolation in cell culture is the standard method for diagnosing HSV infection, with confirmation by specific immunological staining. The performance of the Rapid Absorbent Matrix Pad (RAMP) HSV culture confirmation test was evaluated with specimens obtained from 71 patients with suspected HSV infection and inoculated into African green monkey kidney cell lines. Forty-one culture-positive specimens were confirmed to be HSV by both the RAMP HSV test and the Bartels HSV immunoperoxidase test. Thirty immunoperoxidase-negative specimens were also negative in the RAMP HSV test. The sensitivity and specificity of the RAMP HSV test were 100%. Twelve specimens positive for cytomegalovirus, adenovirus, or enterovirus tested negative by the RAMP HSV test. Thus, the RAMP HSV test was faster than, easier to perform than, as sensitive as, and as specific as other well-documented confirmation methods.