Glycopeptides such as vancomycin are antibiotics of last resort whose biosynthetic pathways still hold undefined details. Chemical probes were used to capture biosynthetic intermediates generated in the nonribosomal peptide formation of vancomycin in vivo. The putative intercepted intermediates were characterised via HR-LC-MS2. These species provided insights into the timing of the first chlorination of the peptide backbone by the halogenase VhaA: this holds significant interest for enzyme engineering towards the making of novel glycopeptides.