Amphibian skin secretions contain abundant bioactive peptides that are valuable natural resources for human beings. However, many amphibians are disappearing from the world, making relevant scientific studies even more important. In this study, 24 cDNA sequences encoding antimicrobial peptide (AMP) precursors were initially cloned by screening a cDNA library derived from the skin of the Sichuan torrent frog, Amolops mantzorum. Eighteen mature AMPs belonging to 11 different families were deduced from these cDNA clones. Biological function was confirmed in each family of these AMPs. Some of them were purified from the skin secretions, and their molecular structures were determined by Edman degradation. Liquid chromatography in conjunction with tandem mass spectrometry (LC-MS/MS)-based peptidomics was used to further confirm the actual presence and characteristics of mature AMPs in the skin secretions of A. mantzorum. Incomplete tryptic digestion and gas-phase fractionation (GPF) analysis were used to increase the peptidome coverage and reproducibility of peptide ion selection.