Embodiments described herein relate to assay methods and kits for detecting resistance of any enzyme to its inhibitor due to functional alteration of the enzyme, comprising, conducting two or more reactions with two or more reagent mixes optionally containing substrates for the enzyme. The mixes are substantially similar, except that one contains no enzyme inhibitor whereas the others contain an enzyme inhibitor being tested for resistance. The ratio of the signal from the reaction with an inhibitor to that from a reaction without an inhibitor is used to indicate whether the enzyme is resistant to the enzyme inhibitor and also determine the susceptibility or resistance of the enzyme to various inhibitors and further identify enzyme variants. Embodiments further relate to assay methods comprising only two reactions—one conducted in a mix containing the inhibitor and the other without the inhibitor. Further included are devices for conducting such assays.