A facile method for the quick discovery and quantification of isonitrile compounds from microbial cultures was established based on the isonitrile-tetrazine click reaction. This method was successfully applied to the rediscovery of diisonitrile antibotic SF2768 from an unknown strain Streptomyces tsukubensis. Finally, an in situ reduction further enabled bioorthogonal ligation of primary and secondary isonitriles for the first time.