Proteases play a critical role in many of the physiologic processes of wound repair. However, if their activity becomes uncontrolled proteases can mediate devastating tissue damage and consequently they have been implicated in chronic wound pathophysiology. Previous studies have shown that chronic wound fluid contains elevated protease levels that have deleterious effects, degrading de novo granulation tissue and endogenous biologically active proteins such as growth factors and cytokines. Therefore, we have proposed that an effective therapeutic approach for chronic wounds would be to modify this hostile environment and redress this proteolytic imbalance. Using an ex vivo wound fluid model, we show the ability of a proprietary new wound treatment to bind and inactivate proteases. We have shown that the addition of this test material to human chronic wound fluid obtained from diabetic foot ulcer patients resulted in a significant reduction in the activities of neutrophil-derived elastase, plasmin, and matrix metalloproteinase when compared to wet gauze. This study provides mechanistic evidence to support the hypothesis that this novel treatment modality for chronic wounds physically modifies the wound microenvironment, and thereby promotes granulation tissue formation and stimulates wound repair.