Damage to the intestinal epithelial barrier during hemorrhagic shock results in tissue destruction and increased permeability. Although the downstream effects of damage to the intestinal epithelial barrier have been investigated, the mechanism behind the increase in permeability has not been elucidated. Previous studies have suggested a proteolytic source for the degradation, but the spectrum of proteolytic enzymes in the intestinal tissue is very broad. We employed mass‐spectroscopy based proteomic analysis of a jejunal intestinal sample obtained from a young adult male Wistar rat (flushed with PBS to remove luminal contents) and found that the enzymes with the greatest relative abundance among the proteolytic enzymes were cathepsin–B, –C, and –Z. We examined cathepsin localization profiles using rat tissue from normal control (NC) Wistar rats and rats that underwent ischemia (at a blood pressure of 35 mmHg) followed by 2 hours of reperfusion (IR). Immuno‐histochemistry (IHC) was used to determine the cathepsin localization profiles. In the case of NC, comparison of cathepsin–B, –C, and –Z localization profiles of the intestinal epithelial barrier between rats (in our study) and humans (Swedish Human Protein Atlas) revealed a significant degree of similarity. Western Blots and gel zymography were conducted to determine changes in cathepsin concentration and activity respectively. Western blots showed a decrease in cathepsin levels, while gel zymography showed no changes in cathepsin activity between NC and IR groups. However, cathepsin activity per mole of total cathepsin (expressed by the ratio between the gel zymography and western blot bands) was higher in the case of IR, supporting the potential role cathepsins play in causing damage to intestinal tissue during hemorrhagic shock, leading to increased intestinal permeability.Support or Funding InformationSupported by HL 10881.