In situ click chemistry has great potential for identifying enzyme inhibitors. However, conventional in situ click chemistry provides extremely low yields of the products, making it incompatible with direct activity-based assays. Here, to address this issue, we focused on the catalysis of azide-alkyne cycloaddition (AAC) by the metal ion in metalloproteins. We chose 2-ethynyl N-heterocompounds as alkyne fragments which are activated by coordination to the metal ion. For proof of concept, we applied metal ion-catalyzed in situ AAC to identify inhibitors of Fe(II)-dependent lysine demethylase 5C (KDM5C). The triazole product was obtained in dramatically high yield, dependently on Fe(II) in KDM5C, and the metalloprotein-catalyzed click reaction was compatible with activity-based high-throughput screening, enabling us to discover a potent KDM5C inhibitor. Thus, metal-catalyzed in situ AAC should be generally applicable to other metalloproteins.