Several factors reduce diagnosis of human immunodeficiency virus (HIV) in infancy in resource-limited settings. Simple, rapid antibody assays cannot be used because of the presence of maternal antibodies. As a consequence, viral nucleic acids or proteins must be detected. Nucleic acid assays are technologically complex and subject to contamination and require expensive equipment and highly trained technologists, limiting availability to selected centers of excellence in resource-limited countries. The ultrasensitive p24 antigen assay (Up24), an alternative that overcomes the limitations of antibody assays while using an enzyme-linked immunosorbent assay (ELISA)-based platform, reduces the need for expensive equipment and personnel. This study will examine parameters of use of the assay, exploring alternative markers.