The Centers for Disease Control and Prevention recommends nucleic acid amplification testing for chlamydia and gonorrhea in sexually abused girls. No studies describe performance of APTIMA Combo 2 Assay with second target confirmation on the same testing platform. This nucleic acid amplification testing is evaluated within a large child advocacy center. Girls 3 to 18 years, 35% of whom reported consensual sexual activity, were prospectively tested by APTIMA Combo 2 on urine/vaginal swabs and by vaginal culture. A case of infection was defined as positive culture or positive urine or vaginal swab nucleic acid amplification testing with second target confirmation. Sensitivity of APTIMA Combo 2 on urine was found to be superior to vaginal culture and comparable to APTIMA Combo 2 on vaginal swabs for both infections. APTIMA Combo 2 on urine is less invasive, and its use may be preferred in this traumatized population. (Contains 3 tables.)