To directly identify protein off-targets of Ciprofloxacin and Levofloxacin, we designed chemical probes suitable for in situ protein target identification studies via AfBPP (Figure ). In order to enrich and identify non-covalently binding proteins, a diazirine photo-crosslinker together with an alkyne tag was incorporated in the core FQ-scaffold. The piperazine ring, present in both drugs, was selected for modification with the minimal photo-crosslinker as previous structure-activity relationship studies (SAR) for antibiotic activity in bacteria indicated flexibility in the modification of the amine at position C4. Alternatively, we modified the acid moiety with a photo-crosslinker to account for a putative different binding mode of human protein off-targets and maximize the chances of their identification. Appending the photo-crosslinker at the carboxylic acid also impairs the well-established bivalent metal complexation of FQs and thus could help to decipher off-targets unrelated to metal binding.