Protease stimulation in HEK due to mustard (sulfur mustard, SM; nitrogen mustard, NM) exposure is well established. However, the specific protease(s) stimulated by mustard and the protease substrates remain to be determined. In this study, we observed that mustard stimulates several proteases, and the epidermal-dermal attachment protein laminin-5 is one of the substrates. In mustard-exposed skin, laminin-5 degradation results in the detachment of the epidermis from the dermis and, therefore, vesication. We utilized gelatin zymography, Western blotting, and immuno-fluorescence staining techniques to study the mustard-stimulated proteases and laminin-5 degradation in HEK. Two major protease bands (NM: 64 kDa and SM: 72 kDa) were observed by zymography in mustard-exposed cells. Addition of serine protease inhibitors (50 μM ICD 2812 or 1 mM PMSF), the metalloprotease inhibitor 1, 10-phenanthroline (1 mM), or the caspase inhibitor Z-VAD-FMK (10 μM) to cells prior to SM exposure decreased the mustard-stimulated protease bands (zymography), and completely prevented mustard-induced laminin-5 degradation (Western blotting, immuno-fluorescence). In conclusion, our results in the HEK model indicate that (a) mustard stimulates multiple proteases in the skin, and (b) protease inhibitors are prospective vesicant countermeasures.