Background The Histoplasma urine antigen (HUAg) is the preferred method to diagnose progressive disseminated histoplasmosis (PDH) in HIV patients. In 2007, IMMY ALPHA Histoplasma EIA was approved for clinical for on-site use, and therefore useful for regions outside the United States. However, ALPHA-HUAg is considered inferior to the MVista-HUAg which is only available on referral. We aim to evaluate the diagnostic accuracy of ALPHA-HUAg. Methodology/Principal findings We conducted a multicenter, prospective, diagnostic test study in two secondary and eight tertiary-care facilities in Mexico. We included HIV patient with PDH suspicion and evaluated ALPHA-HUAg diagnostic accuracy using as reference standard the Histoplasma capsulatum growth on blood, bone marrow, and tissue cultures or compatible histopathologic exam (PDH–proven). We evaluated the results of 288 patients, 29.5% (85/288; 95% confidence interval [CI], 24.3–35.1) had PDH. The sensitivity of ALPHA-HUAg was 67.1% (95% CI, 56–76.8%) and the specificity was 97.5% (95% CI, 94.3%-99.1%). The positive likelihood ratio was 27.2 (95% CI; 11.6–74.4). In 10.5% of the PDH–proven patients, a co-existing opportunistic infection was diagnosed, mostly disseminated Mycobacterium avium complex infection. Conclusions/Significance We observed a high specificity but low sensitivity of IMMY-HUAg. The test may be useful to start early antifungals, but a culture-based approach is necessary since co-infections are frequent and a negative IMMY-HUAg result does not rule out PDH.