Many drugs are inhibitors of enzymes involved in mediating the disease processes. Understanding the mechanism of action (MOA) of the target enzyme is critical in early discovery and development of drug candidates through extensive Structure-Activity Relationship (SAR) studies. This chapter contains a primer on the MOA of enzymes and its significance in drug discovery, types of inhibition, development and validation of MOA assays, data analysis and guidelines for performing these assays. New and experienced investigators will find this chapter useful when starting new projects involving enzyme targets.