Paper-based diagnostic devices have been widely applied to assess the presence and status of a variety of clinical diseases by analyzing samples such as urine or blood. Due to their low cost, user-friendliness, and convenience, they have been used as point-of-care (POC) devices in countries lacking resources or energy. Despite wide-ranging research and implementation, paper-based devices have not previously been developed for wound analysis. Here, we discuss the successful development of such a tool to facilitate simple and rapid wound status assessment. The purpose of this study was to develop a paper-based elastase detection device (PEDD) for clinical wound assessment that specifically examines human neutrophil elastase (HNE), one of the most abundant serine proteases found in chronic wounds. The first step in this study was an examination of different paper substrate types (i.e., chromatography paper and filter paper) to determine which provided the best protease immobilization and colorimetric response. We then used a wax printing approach to create hydrophobic and hydrophilic regions and designated test zones created on both chromatography and filter papers. This allowed us to physically immobilize both substrate and protease within the desired test zone regions. This PEDD which demonstrated good sensitivity (0.631 μg mL-1, in a wound fluid system) can be used to monitor protease activity expressed in wounds. After developing this device, we examined samples from 9 patients presenting a total of 7 acute and 4 chronic wounds to determine wound HNE concentration. We believe that this study may be widely applicable in both academic and commercial sciences, including the development of practical POC detection devices.