The effect of treatments with four herbicides and a safener on the activity of triosephosphate isomerase (TPI) extracted from shoots of Italian ryegrass was investigated. It was found that atrazine and fluorodifen, herbicides which interfere with photosynthesis, caused a decrease in measured enzyme activity. In addition, the in vitro effect of oxidized glutathione (GSSG), a compound produced in situations of oxidative stress, on TPI activity was investigated. It was shown that GSSG was a strong inhibitor of enzyme activity, at low concentrations in a dose-time-dependent manner. The enzyme extracts were submitted to chromatographic purifications and to two-dimensional electrophoresis. Some spots had molecular masses ranging between 20 and 30 kDa and were characterized and identified by LC-ESI-MS/MS as TPIs. The mass spectrometry also made it possible to identify the presence of cysteine residues that could be subjected to S-glutathionylation, which regulate the enzyme activity.