It is well established that sexually acquired genital ulcer disease (GUD) facilitate the heterosexual transmission of HIV infection. Thus, prompt diagnosis and treatment of GUD is important not only to reduce morbidity but also to limit the transmission of HIV infection. To diagnose the etiology of GUD based solely on clinical grounds is not very reliable, especially among HIV-infected individuals. In addition, the most common cause of GUD can vary geographically and temporally. Molecular diagnosis of multiple GUD etiologies simultaneously using the real-time multiplex PCR assay provides a rapid, sensitive, accurate, and cost effective alternative than standard laboratory tests.