Laboratory diagnosis of microbial agents associated with sexually transmitted infections plays an important role in both the care of victims of child sexual abuse (CSA) and the investigation of suspected CSA incidents, with law enforcement implications. Rapid and sensitive test results prompt immediate actions to treat and protect the victimized children. The development and maturation of automated nucleic acid amplification tests (NAATs) has greatly improved the assay sensitivity and specificity, with only a 1- to 2-h turnaround time. Unfortunately, the performance characteristics of NAATs have been determined largely with a few limited specimen types and evaluated in adults only. This minireview attempts to cover the scope of infectious agents potentially implicated in CSA, specimen collection, laboratory test modalities, and laboratory report constraints, further complicated by infrequently collected specimen types from prepubertal children <13 years of age.