In this study, we used mass spectrometry-based discovery proteomic analysis to examine the cell-wide changes of Mycobacterium smegmatis, in response to sub-lethal concentrations of H2O2 and nitric oxide (NO) over time. Preliminary results have shown that sub-lethal concentrations of reactive oxygen and nitrogen species may act as signalling molecules by playing a role in the overall protein abundance that may be conducive to survival in vivo. The presented study showed that both treatments induced: the DosR regulon, alterations in protein expression and lipid metabolism. In addition to the proteomic changes observed, we have also observed increased survival after macrophage infection of Mycobacterium smegmatis pre-exposed to sub-lethal doses of H2O2 or DETA-NO. ¬ In total, 27 samples yielded approximately 3300 M. smegmatis protein group identifications. More in-depth analysis of this new dataset will provide a better understanding as to the inner workings of mycobacteria under oxidative and nitrosative stress and potential mechanisms by which exposure to sub-lethal concentrations of stressors results in an increased resistance to stress.