The Huisgen cycloaddition reaction of an alkyne with an azide to give a 1,4-disubstituted-1,2,3-triazole, is catalysed by copper(I) in what is an example of a click reaction. There are also limited reports on using an enzyme to catalyse this reaction, most notably employing a library of azides and/or alkynes in order to screen and selectively synthesize potent inhibitors of the target enzyme. Here the enzyme functions as a template to ‘select’ the optimum combination of azide and alkyne fragments. When these are correctly positioned in the active site, an irreversible 1,3-dipolar cycloaddition reaction occurs to produce the corresponding triazole. Paradoxically, the ability to produce high-affinity inhibitors is also a potential limitation of this methodology. Tight binding inhibitors often remain associated in the protein’s active site, thereby preventing multiple rounds of catalysis. New approaches that increase product yields, and hence improve the sensitivity of detection, would enable this example of template-guided synthesis to become more widely employed in drug discovery. Here we present an approach for improving the detection of triazole products generated by in situ click chemistry that uses an appropriately engineered protein variant of biotin protein ligase from Staphylococcus aureus¹. The enzyme is an ideal for in situ click chemistry as it contains two adjacent binding pockets for ligands biotin and ATP². A mutant enzyme was designed to increase the dissociation of ligands from the active site, thereby facilitating multiple cycles of the Huisgen cycloaddition reaction. We demonstrated that the enzyme itself was capable of synthesising chemical analogues of the reaction intermediate from a small library of precursors¹. Moreover, the mutant enzyme yielded high quantities of the triazole, thereby improving the detection of the products by HPLC and LC-MS. We propose that this approach could similarly be applied to other drug targets for the discovery of enzyme inhibitors.