The Acquired Immune Deficiency Syndrome (AIDS) affects the life of millions of people around the world. Although rapid and low cost screening tests are widely available for the diagnosis of HIV infection, the count of CD4+ T lymphocytes remains a drawback in the areas mostly affected by the HIV, being this control imperative for assessing the deterioration of the immunological system and the progression towards AIDS, when the counting of cells falls down 200cellsμL−1. This paper describes a high-throughput, simple and rapid method for CD4+ T lymphocytes quantification, directly in whole blood, based on a magneto ELISA. The CD4 cells are separated and preconcentrated from whole blood in magnetic particles, and labeled with an enzyme for the optical readout performed with a standard microplate reader. The magneto ELISA is able to reach the whole CD4 counting range of medical interest, being the limit of detection as low as 50 CD4+ cells per μL of whole blood, without any pretreatment. This method is a highly suitable alternative diagnostic tool for the expensive flow cytometry at the community and primary care level, providing a sensitive method but by using instrumentation widely available in low-resource settings laboratories and requiring low-maintenance, as is the case of a microplate reader operated by filters. Display omitted •CD4 magneto immunoassay for whole blood with a LOD of 50CD4cellsμL−1.•The test spans the clinical range for its application in AIDS diagnosis and following up.•The CD4 cells are preconcentrated from whole blood without any sample treatment.•The magneto immunoassay offers an alternative to flow cytometry in low resource settings.•This high-throughput test allows the detection of several samples in decentralized small health centers.