Enzymatic assays for HTS are configured to suitably aid the identification of orthosteric and allosteric modulators. Beside playing a central role as primary assays for HTS campaigns and Hit-to-Lead activities, enzymatic assays are highly suitable as secondary assays to address compound interference, selectivity, and specificity, as well as to provide insights on the mechanism of action of test compounds. We apply functional enzymology using HTS-grade assays to investigate whether compounds are reversibly or irreversibly affecting the target enzyme, and if their binding is competitive, non-competitive, or uncompetitive with the substrate. Moreover, key parameters such as association and dissociation kinetics between targeting compounds and enzyme are determined while the enzyme is accomplishing its catalytic reaction, which translates into more meaningful data for hit to lead progression of novel chemical series.