A simple diagnostic method for mechanistic analysis of reversible enzyme inhibitors is presented. The method involves simple experimentation to determine how the inhibition by a reversible inhibitor changes in response to the substrate concentration varied in the assay. Four types of inhibitors are categorized based on their kinetic characteristic: (1) competitive or mutually exclusive inhibitors that compete with the substrate for the enzyme; (2) noncompetitive inhibitors that are independent of the substrate for binding to the enzyme; (3) antagonistic inhibitors where the binding affinity of the inhibitor is partially reduced by the substrate binding to the enzyme; and (4) synergistic inhibitors where inhibitor binding is enhanced by substrate binding. An equation was derived for data fitting and subsequent determination of inhibitor binding mode. The method was evaluated in three model enzyme systems, i.e., PK, adenylate kinase, and LDH, with known inhibitors. The method was also used to characterize a large number of unknown Csp3 inhibitors identified from HTS of a compound library consisting of 120,000 distinct chemical entities, a field test that validated the utility of the method. Among 76 Csp3 inhibitors analyzed, 70 were found to be non-mutually exclusive inhibitors, suggesting the existence of an allosteric site(s) in Csp3 for effective inhibition. The implication of this observation is discussed.