Diagnostic strategies for HIV infection have evolved to keep pace with the development of reliable and sensitive laboratory methods. FDA-approved fourth-generation antigen (Ag)-antibody immunoassays afford improved diagnosis but can result in interpretive challenges when used in unique patient populations, including infants, pregnant women, the setting of post-exposure prophylaxis, and CAR-T cell ... [Show full abstract] recipients. The ordering and reporting challenges for serological and molecular methods are further complicated by the availability of a CLIA-waived rapid test platform, as well as a fifth-generation immunoassay that differentiates HIV types simultaneously with detection. However, these assays do not precisely align with the current CDC/Association of Public Health Laboratories (APHL) guidelines. Finally, the notable absence of test platforms for FDA-approved qualitative HIV-1 RNA and integrated proviral DNA detection impacts the clinical and diagnostic approach. Here, we describe challenges and opportunities regarding the diagnosis of HIV infection in both routine and unique patient populations.