Mycothiol (MSH) is the principal low-molecular-weight thiol, unique to mycobacteria and other actinomycetes, that performs a role analogous to glutathione found in other organisms. MSH plays a key role in oxidative stress management and is oxidized to the dimeric mycothiol disulfide (MSSM) in the process. NADPH-dependent mycothiol disulfide reductase (Mtr) helps to maintain an intracellular reducing environment by reducing MSSM back to MSH. Mtr inhibition studies are currently impaired by limited availability of MSSM. Substrate demands are particularly high in time-dependent inhibition assays. Here we report an assay that chemically recycles a mixed disulfide substrate analogue in situ, thereby greatly reducing the substrate quantities needed for such assays. This has enabled the development of a continuous assay where linear reaction rates can be maintained for 40 min or longer using minimal substrate concentrations (5 microM versus a substrate K(m) value of 268 microM). In this manner, substrate requirements are reduced by orders of magnitude. Characterization of a novel time-dependent inhibitor, 2-(5-bromo-2-methoxyphenyl)acrylonitrile, is also demonstrated using these procedures.