The current demand for HIV diagnostic tests in resource-limited settings is not being fully addressed by the currently available nucleic acid test or enzyme-linked immunosorbent assay-based p24 antigen assays. This is primarily due to their high cost, their requirement for controlled laboratory conditions and/or personnel, or the relatively long turn-around time for test results. Self performing lateral flow assays address all of these issues but to date have not reached the level of analytical sensitivity for HIV p24 demonstrated by current generation enzyme-linked immunosorbent assay technology. This report presents an initial prototype lateral flow assay for HIV p24 antigen capable of achieving subpicogram per milliliter limits of analytical sensitivity and requiring less than 40 minutes to yield results.