There are few standards for the clinical examination methods used in suspected cases of sexual abuse; therefore, there is debate about which laboratory test methods should be used. The lack of standards for sample collection from and testing of children creates additional challenges, in part because of the differences in biology and pathogen interaction with children's cells and in part because of the additional emotional impact of a pediatric case. The incidence of child sexual abuse (CSA) is much higher than the general population or health care professionals realize. Potential CSA victims have been traumatized by the experience; therefore, the physical examination that follows needs to be performed in a “safe” environment. The laboratory may get requests to evaluate samples collected from potential perpetrators, as well. The normal pre-analytical, analytical, and post-analytical procedures used are inadequate when used to evaluate specimens collected from survivors of abuse. Specific procedures and policies need to be instituted to aid in the diagnosis of a sexually transmissible disease (STD), indicative of sexual contact. Coordinating the examination of potential victims requires using a multidisciplinary team to ensure proper collection, delivery, and processing of samples, and the identification of organisms known to cause an STD is a difficult task. In addition, the results of testing and potential confirmation of positive results are important aspects of care. The original sample must be retained so that additional testing may be performed, if indicated. Since many potential perpetrators admit to the crime following positive STD organism identification, the microbiology laboratory must work with clinicians, nursing, law enforcement, administrators, and the legal department of the health care system to offer testing for STDs. Approved interdisciplinary policies should be stringently followed, and the laboratory must be ready to be cross-examined during the litigation process. Practical suggestions for those performing testing for CSA are detailed in this review.