A large number of diverse antimicrobial peptides have been found in amphibian skins, and many more remain to be identified. It is sufficiently easy to obtain amounts of gland secretions sufficient for both identification and functional testing of the bioactive peptides. We describe here a systematic peptidomics approach which we combined with genomics and functional testing. This has proven to be an effective way to identify amphibian antimicrobial peptides, including novel peptide families. Protocols are exemplified for Bombina maxima and Odorrana grahami and can be easily adapted for use with other amphibian species.