The difficulty of diagnosing HIV in infants is a major obstacle to early antiretroviral therapy (ART) in resource-limited settings. As serological tests are unreliable during the first 18 months of life, and the cost and complexity of polymerase chain reaction (PCR)-based assays limit their access in resource-limited settings, p24 antigen detection has emerged as an alternative diagnostic tool. In this study, the performance of an ultrasensitive p24 antigen assay on dried blood spots was evaluated under field conditions in rural Tanzania. Specimens were stored and shipped at tropical room temperature, and analysed within six weeks. In total, 27 consecutive children aged <18 months and exposed to vertical HIV transmission were enrolled. Overall sensitivity and specificity was 100% (95% confidence interval [CI], 47.8-100) and 95.5% (95% CI, 77.2-99.9), respectively. Our findings suggest that detection of p24 antigen on dried blood spots can be a reliable and feasible diagnostic tool for infant HIV infection in rural resource-limited settings.