In recent decades, the field of molecular diagnostics for human immunodeficiency virus (HIV)/ acquired immunodeficiency syndrome (AIDS) has seen significant progress, revolutionizing the detection and management of HIV infection. Techniques like polymerase chain reaction (PCR) and nucleic acid amplification tests (NAATs), have greatly improved the accuracy and sensitivity of HIV detection. These techniques can identify the presence of the virus even during the window period, which is the early phase of infection when antibodies have not yet developed and traditional antibody tests might fail to detect the virus. Molecular assays are essential for monitoring viral load, detecting drug resistance, mutations, guiding treatment decisions, and improving patient care in HIV management. This chapter underscores the pivotal role of molecular diagnostics in the global efforts to combat HIV/AIDS. By enabling timely and precise diagnosis, facilitating effective treatment strategies, and improving patient outcomes, these techniques are vital in addressing the HIV epidemic. The advancements in molecular diagnostics not only enhance the quality of life for those living with HIV but also contribute to broader public health goals, such as reducing transmission rates and moving closer to an AIDS-free generation.