Functional protein analysis offers unique insights into phenotype-related changes that cannot be fully captured through genetic or transcriptomic data alone. This renewed focus has sparked growing interest in activity-based diagnostics. Conventional methodologies treat 10-10 protein molecules in bulk. Meanwhile, new approaches are emerging to probe the functions of individual protein molecules. In this Outlook, we discuss recent challenges in applying single-molecule enzyme activity assays to understand disease-related alterations in enzyme activity landscapes at the proteoform level, with potential applications in disease diagnosis.