Histoplasmosis is a major cause of mortality in people living with HIV (PLHIV). Rapid methods to diagnose Histoplasma capsulatum disease could dramatically decrease the time to initiate treatment, resulting in reduced mortality. The aim of this study was to validate a MiraVista® Diagnostics (MVD) Histoplasma urine antigen lateral flow assay (MVD LFA) for the detection of H. capsulatum antigen (Ag) in urine and compare this LFA against the MVista® Histoplasma Ag quantitative enzyme immunoassays (MVD EIA). We assessed the MVD LFA using a standardized reference panel of urine specimens from Colombia. We tested 100 urine specimens, 26 from PLHIV diagnosed with histoplasmosis, 42 from PLHIV with other infectious diseases, and 32 from non-HIV infected persons without histoplasmosis. Sensitivity and specificity of the MVD LFA was 96%, compared with 96% sensitivity and 77% specificity of the MVD EIA. Concordance analysis between MVD LFA and the MVD EIA displayed an 84% agreement, and a Kappa of 0.656. The MVD LFA evaluated in this study has several advantages, including a turnaround time for results of approximately 40 min, no need for complex laboratory infrastructure or highly trained laboratory personnel, use of urine specimens, and ease of performing.