Many wildlife species including rhinos are susceptible to infection with Mycobacterium tuberculosis or M. bovis. Antemortem diagnostic testing in large exotic hoof stock species has been limited by challenges associated with test administration, sample collection, and interpretation. Hence, a simple, rapid, blood-based test is needed. Two confirmed M. tuberculosis-infected black rhinoceros and one exposed suspect were evaluated for antibody responses using a lateral-flow rapid test (ElephantTB STAT-PAK) and multi-antigen print immunoassay (MAPIA). All three animals were seropositive by both tests. MAPIA detected antibodies to ESAT-6, CFP10, and MPB83 antigens. When the rhinos were treated with antitubercular therapeutics, their antibody responses gradually declined. One rhinoceros died approximately 9 mo after initiation of treatment and showed an increase in antibody titer shortly before death. The other two rhinoceros, which were treated for 1 and 2 yr, respectively, had no clinical signs or positive culture for M. tuberculosis at the time of necropsy performed 2 or 6 yr later for unrelated reasons. The antibody levels in these rhinos continued to be significantly decreased. The findings suggest that the ElephantTB STAT-PAK and MAPIA may be useful tools to detect M. tuberculosis infection and monitor treatment in black rhinoceros.