Mycothiol (MSH), the primary low-molecular weight thiol produced in mycobacteria, acts to protect the cell from oxidative stress and to maintain redox homeostasis, notably in the pathogenic Mycobacterium tuberculosis in the course of human infection. The mycothiol disulfide reductase (Mtr) enzyme reduces the oxidized form of mycothiol, mycothione (MSSM), back to MSH, however its role in bacterial viability is not clear. In this study, we sought to determine the MSH levels of wild-type (WT) and Mtr mutant mycobacteria during oxidative stress. We describe a rapid method for the relative quantification of MSH using high-sensitivity mass spectrometry (MS) with selected ion monitoring (SIM). This method uses only minimal sample cleanup, and does not require advanced chromatographic equipment or fluorescent compounds. MSH levels decreased in the Mtr mutant only upon treatment with peroxide, and the results were consistent between our method and previously-described thiol quantification methods. Our results indicate that our MS-based method is a useful, high-throughput alternative tool for the quantification of MSH from mycobacteria.