When developing enzyme assays for HTS, the integrity of the target enzyme is critical to the quality of the HTS and the actives, or “hits”, identified through the screens. The incorrect identity or lack of enzymatic purity of the enzyme preparation will significantly affect the results of a screen. In this chapter, the authors discuss in detail the consequences of impure and mis-identified enzyme preparations, potential steps that can be taken to avoid measurement of the wrong activity and methods to validate the enzymatic purity of an enzyme preparation.