Cellular activities depend on the continual synthesis, modification, and breakdown of molecules. In this self-sustaining network of chemical activity we refer to as life, proteins organize and catalyze almost every reaction process. Proteolysis is a general process whereby one protein, known as a protease, cuts a peptide bond in another protein. Proteases have been well characterized over the years, however, there remain some proteases where biochemical activities are relatively unknown or may benefit from more detailed studies. Single-molecule nanopore based assays have the advantage of detecting proteins with high specificity and resolution, and in a label-free, real-time fashion. The focus of this collective work has been the development of nanopore methods useful for 1) analyzing the composition of ubiquitin polymers and their regulation by deubiquitinase proteolytic enzymes, and 2) analyzing the conformation dynamics of the West-Nile virus NS2B/NS3 protease. In the process, we also explored strategies for optimization of nanopore-analyte interactions. These nanopore methods developed here have the advantages of being real-time and label-free approaches, which may be applied using high-throughput nanopore arrays to studies of protein and enzyme conformations, activities, and inhibition.