Currently, there are around 150,000 HIV-infected patients in Spain. This number, together with the fact that this disease is now a chronic condition since the introduction of antiretroviral therapy, has generated an increasing demand on the clinical microbiology laboratories in our hospitals. This increase has occurred not only in the diagnosis and treatment of opportunistic diseases, but also in tests related to the diagnosis and therapeutic management of HIV infection. To meet this demand, the Sociedad de Enfermedades Infecciosas y Microbiología Clinica (Spanish Society of Infectious Diseases and Clinical Microbiology) has updated its standard Procedure for the microbiological diagnosis of HIV infection. The main advances related to serological diagnosis, plasma viral load, and detection of resistance to antiretroviral drugs are reviewed in this version of the Procedure.