Updated project metadata. Many pathogenic bacteria utilize posttranslational modifications to manipulate central components of the host cell functions. Often, the released enzymes belong to the large Fic-family, causing target modification with nucleotide monophosphates. Due to the lack of a generic method for the identification of the Fic-family’s cellular targets, we have established a new approach by combining synthetic thiol-reactive nucleotide derivatives (TReNDs) with recombinantly produced Fic-enzymes containing strategically placed cysteines in their active sites, thereby producing reactive binary FicTReND probes for covalent substrate capture. The binary probes successfully capture their targets from complex lysates and permit their identification by mass spectrometry.