Engineering of biosynthetic enzymes is increasingly employed to synthesize structural analogues of antibiotics. Of special interest are non-ribosomal peptide synthetases (NRPSs) responsible for production of important antimicrobial peptides. Here, directed evolution of an adenylation domain of a Pro-specific NRPS module completely switched substrate specificity to the non-standard amino acid piperazic acid (Piz) bearing a labile N-N bond. This success was achieved by UPLC-MS/MS based screening of small, rationally designed mutant libraries and can presumably be replicated with a larger number of substrates and NRPS modules. The evolved NRPS produces a Piz-derived gramicidin S analogue. Thus, we give new impetus to the too-early dismissed idea that widely accessible low-throughput methods can switch the specificity of NRPSs in a biosynthetically useful fashion.