Summary Subtilisin is a proteolytic enzyme with a multitude of industrial uses, including as a degreaser in biological parts washers and for pre-sterilization cleaning in hospitals. Respiratory allergic reactions related to exposure to the proteolytic enzymes found in cleaning products have been reported. It is known that workplace exposure to enzymes causes sensitization that can lead to occupational asthma. The allergenic potential of subtilisin is identical to that of other enzymes—such as amylases, cellulases and lipases. Subtilisin’s threshold limit value-ceiling is very low (60 ng/m3) and recirculation of air from rooms in which it is used is prohibited. However, despite the existence of a ceiling value, there is no standard specific method for measuring the risks of workplace exposure to subtilisin. The main purpose of this study was to develop and implement a specific analytical method for subtilisin at concentrations found in the workplace. Though non-specific, a method for proteolytic enzyme determination was developed. This method can rapidly determine concentrations of all airborne subtilisin-type proteases in the workplace. It can also be used to quantify the protease content of soaps, which will mean fewer air sampling procedures in hospitals that use detergents containing subtilisin. In other words, testing of products used could constitute the first step in the assessment of workplace exposure. Sampling of ambient air in participating hospitals demonstrated that manual washing of instruments and endoscopes is the main source of workplace exposure to aerosols that could contain subtilisin. Automatic washers did not prove to be a source of exposure. A secondary objective of the study was to explore the possibility of proteomic characterization of proteins, subtilisin in particular, in the soaps used in the participating hospitals. Proteomic analyses were used to characterize the proteins in the soaps and the presence not only of subtilisin but also of other enzymes, such as alpha-amylases, was confirmed. Proteomic analysis for occupational health and safety (OHS) purposes seems to have real potential for characterization of enzymes in the workplace and should be explored. Substitution as a preventive measure is definitely an approach to consider, as subtilisin-free products are gradually becoming available, but caution is in order. As our research on proteomic analysis demonstrates, soaps may contain other sensitizing enzymes, and this must be taken into consideration and verified.