Individuals with acute HIV infection (AHI) pose a greater transmission risk than most chronically HIV-infected patients and prevention efforts targeting these individuals are important for reducing the spread of HIV infection. Rapid and accurate diagnosis of AHI is crucial. Since symptoms of AHI are nonspecific, its diagnosis requires a high index of suspicion and appropriate HIV laboratory tests. However, even 30 years after the start of the HIV epidemic, laboratory tools remain imperfect and only a few individuals with AHI are identified. We review the clinical presentation of the acute retroviral syndrome, the laboratory markers and their detection methods, and propose an algorithm for the laboratory diagnosis of AHI.