Enzyme assays are standardized experimental protocols, which are established in order to measure the activity or concentration of enzymes in biochemical or cell-based systems. Mostly enzymatic assays are based upon the detection of fluorescent, luminescent, or spectrophotometric endpoint signal. During recent years, florescence-based, absorbance-based, and luminescence-based high-throughput screening assays have been developed and widely used in various fields. Enzyme assays represent an important tool for screening of sample libraries in context of drug discovery. In addition, enzyme assays are valuable for diagnosis of several diseases in clinical settings due to their high sensitivity, specificity, and rapid response. In this chapter, we aim to provide an understanding of the working principle of enzymatic assays and their applications with some specific examples of enzyme assays that are used for drug discovery and diagnostic purpose.