The diagnosis of HIV-1 infection in infants and children continues to present challenges. Currently available virologic assays are sensitive and specific and allow early detection of perinatally acquired HIV infection. Identification soon after birth allows for the rapid initiation of antiretroviral therapy and preservation of the infant's immune system. Serologic diagnostic methods, including HIV-ELISA, Western blot, and immunofluorescence Assay can be used to make the diagnosis of HIV infection in infants older than 18 months of age, children, and adolescents. Recently developed rapid tests allow for testing outside clinical sites, provide results in a short period of time, and allow for prompt initiation of effective prophylaxis in cases of exposure particularly maternal to child transmission. We discuss here the diagnostic management of HIV-exposed infants and HIV-infected children.