Plants have evolved various defense strategies to counter the constant threat from predators and infectious organisms. Aside from physical deterrents such as thorns, plants produce a chemical arsenal consisting of small molecules typically assembled from isoprene units, alongside a range of defense peptides. Cyclotides, found in a range of plant species are ribosomally synthesised defense peptides defined by their unique combination of a cyclic peptide backbone and cystine-knotted topology.¹ Challenges faced in the proteomic analysis of cyclotides stem from difficulties in their chromatographic separation, the necessity for linearisation prior to MS analysis, and their typically poor fragmentation. Here we examine the benefits of orthogonal separation and chemical derivatisation prior to tandem MS in the proteomic characterisation of cyclotides.