Identifying new enzymes and gaining insights into their mode of action (and inhibition) is a critical step in drug discovery. In this chapter, we describe a novel methodology that accelerates this process by combining the precision of activity-based protein profiling (ABPP) with the efficiency of microplate assay technology. Central to this approach is the use of competitive ABPP, which utilizes an activity-based probe (ABP) to directly observe and characterize the interactions between inhibitors and an enzyme. In this study, we use an in-house fluorophosphonate (FP)-based probe, as an example of an electrophilic probe, and pig liver esterase (PLE), as a model enzyme. Simple chemical modification of PLE to introduce a streptavidin tag enables its immobilization and presentation on a biotinylated assay plate. Subsequent competitive ABPP experiments using FP probes and a commercially derived compound library can be run in parallel. This setup allows for the identification of new inhibitors and the estimation of IC50 values in a single operation. This chapter provides a comprehensive protocol for the synthesis of ABPP probes, preparation of enzyme solutions, and execution of plate-based assays. It offers a robust platform for discovering and characterizing novel inhibitors of enzymes with unknown structures or substrate specificities. By applying this technique, we not only expedite the identification of potential drug candidates but also gain deeper insights into their modes of action, thereby enhancing our understanding of their therapeutic potential.