A microtiter alamarBlue assay was adapted and optimized for Mycobacterium avium subsp. paratuberculosis. Using cell concentrations ranging from 10(4) to 10(8) CFU/ml, a minimum incubation time to indicate viability was obtained after 24 h. Rifampin (rifampicin) was used to demonstrate that this method has applications for high-throughput screening against M. avium subsp. paratuberculosis.