Covering: Up to July 2020Ribosomal antimicrobial peptide (AMP) natural products, also known as ribosomally synthesized and post-translationally modified peptides (RiPPs) or host defense peptides, demonstrate potent bioactivities and impressive complexity that complicate molecular and biological characterization. Tandem mass spectrometry (MS) has rapidly accelerated bioactive peptide sequencing efforts, yet standard workflows insufficiently address intrinsic AMP diversity. Herein, orthogonal approaches to accelerate comprehensive and accurate molecular characterization without the need for prior isolation are reviewed. Chemical derivatization, proteolysis (enzymatic and chemical cleavage), multistage MS fragmentation, and separation (liquid chromatography and ion mobility) strategies can provide complementary amino acid composition and post-translational modification data to constrain sequence solutions. Examination of two complex case studies, gomesin and styelin D, highlights the practical implementation of the proposed approaches. Finally, we emphasize the importance of heterogeneous AMP peptidoforms that confer varying biological function, an area that warrants significant further development.