Hydrolases, proteins that cleave covalent bonds using water, represent one of the largest classes of enzymes in the human body and play essential roles in numerous physiological pathways ranging from digestion to nervous system signaling. Despite the prevalence and importance of this enzyme class, the biological functions of many hydrolases remain poorly characterized. Among these less studied members, rhomboid intramembrane proteases (RIPs), which perform hydrolysis chemistry within the hydrophobic environment of biological membranes, are of particular interest. Mutation and dysregulation of these enzymes in neurodegenerative diseases and multiple types of cancer motivates the development of an enhanced understanding of their physiological roles and an investigation of their potential to serve as therapeutic targets; however, methods to conduct these studies are currently lacking. Activity-based protein profiling (ABPP) technology, in which active site-directed chemical probes are used to detect enzyme activity, has facilitated the characterization of many serine hydrolases. As members of this superfamily of enzymes, RIPs represent promising candidates for study by ABPP methods, which can then be leveraged to discover selective inhibitors and to characterize the active proteoforms of these enzymes. This proposal aims to (1) develop optimized ABPP assays to monitor the activity of each of the five human rhomboid proteases in complex biological samples, (2) generate and screen a library of N-sulfonylated heterocycles as potential inhibitors for these enzymes, and (3) characterize proteolytic processing of the ER-localized RIP RHBDL4 and the impact of processing on enzymatic activity. These studies will empower efforts to investigate the physiological roles of the human RIPs and their potential as therapeutic targets. Further, the proposed project will provide an opportunity to train Oberlin undergraduate researchers in contemporary chemical biology methods that incorporate techniques from synthetic chemistry, biochemistry, and molecular biology. Research students at Oberlin will learn theory and advanced laboratory skills not otherwise covered in an undergraduate classroom setting while developing their ability to analyze and present scientific data. Collectively, these activities will enrich the research environment at Oberlin College and help prepare the next generation of researchers in interdisciplinary molecular science.