BACKGROUND There is a worldwide need for a pediatric HIV-1 diagnostic test that has a high diagnostic accuracy, is technically simple and cost efficient. The Up24 HIV-1 assay, which requires both the HIV-1 p24 ELISA and the ELAST signal amplification kit, has previously been shown to be a robust tool to diagnose pediatric HIV-1 from dried whole blood spots (DBS) (Cachafeiro et al., JCM 2009;47:459-62(13)). In order to make the assay more accessible to a resource-limited clinical setting, we eliminated the ELAST system, which simplified the Up24 assay, reduced its cost, and tested the accuracy of the modified assay in a rural Malawian hospital. OBJECTIVE In this proof of concept study, we tested the ability of a simplified Up24 antigen assay, without ELAST, to detect HIV-1 on DBS obtained via heel prick from 6-week-old Malawian infants. METHODS A case-control study of DBS collected from 113 HIV-infected and 109 HIV-negative infants, using the HIV-1 DNA PCR assay as the reference standard. RESULTS The simplified HIV-1 Up24 assay had a sensitivity and specificity of 84% and 98%, respectively. When HIV-1 prevalence is 15%, the positive- and negative-predictive values are 89% and 97%, respectively. CONCLUSIONS The simplified Up24 assay has a good positive- and a robust negative-predictive values, is easier to perform and has a reduced cost compared to both HIV DNA PCR and Up24 assays. With additional testing, the simplified Up24 assay has the potential to increase global access to pediatric HIV-1 diagnostics.