This study explored the role of peroxiredoxin 6 (Prdx6) non-selenium glutathione peroxidase (NSGPx) activity on the regulation of beef tenderization during post-mortem aging periods of up to 168 h. Shear force, NSGPx activity, differential protein expression, heat shock protein 70 (HSP70), heat shock protein 27 (HSP27), and troponin-T levels were quantified for samples from 6 beef longissimum lumborum muscles (LL) that had been treated with either hydrogen peroxide (H2O2), N-acetylcysteine (NAC), mercaptosuccinic acid (MA), or a saline solution (control). In comparison to NAC treatment, MA treatment exhibited inhibitory effects on NSGPx activity and accelerated the tenderization of aged beef within 168 h post-mortem. Proteomics techniques were used to analyze the differential proteins between the treatment groups, comparing samples after 0 and 24 h of post-mortem aging using functional enrichment analyses. These demonstrated the differential proteins to be significantly related to tenderness and primarily via cytoskeleton, extracellular matrix, amino acid metabolism, and apoptosis pathways. After conducting further investigations into the effects of treatment groups on HSP70, HSP27, and troponin-T concentrations in aged beef, it was observed that MA treatment resulted in an increase in the degradation of HSP70, heightened oxidative stress levels, and accelerated degradation of troponin-T. Furthermore, only after 6-12 h post-mortem did the application of H2O2 treatment led to an upregulation in the expression of both HSP70 and HSP27. These findings confirm how oxidative stress treatments affect beef protein expression during post-mortem aging, providing a basis for improving tenderness.