Project 1: Urothelial Cancer of the Bladder (UCB) has an average age of diagnosis nearing 73 years old and has an extremely high rate of recurrence, thus requiring patients to undergo continual testing and monitoring. This contributes to UCB having the highest economic burden of all cancers, costing nearly $4 billion per year. Using multiplex substrate profiling- mass spectrometry and fluorogenic substrate assays a baseline of proteolytic activity in urine in acidic to neutral pH conditions was established. This baseline will prove vital to the discovery of a novel protease biomarker for the use of an inexpensive noninvasive diagnostic for UCB. Project 2: The inhibitor MG132 is a commonly used inhibitor of the proteasome. We determined that this inhibitor is also very potent against the aspartyl proteases Cathepsin D and Cathepsin E. Dose-response curves were generated to show the true specificity of MG132 and its ability to inhibit numerous proteases from different classes. Project 3: Proteases are predicted to function in wound healing however there is little understanding about the role of individual proteases in this process. In order to understand the role of proteases at different stages in the wound healing process, fluorogenic substrate assays were performed. Each substrate was chosen for its ability to capture a specific class of proteases. In order to understand the role of natural killer cells in the wound healing process, wound fluid from NK depleted mice were tested in parallel to an isotope control. Project 4: In order to showcase the role at which proteases play in disease progression a literature review on cyst fluid and peritoneal fluid was conducted. Protease activity in pancreatic, breast and ovarian cysts were evaluated further for its involvement in malignant cyst progression.