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Peptides in Venenum Bufonis remain unexplored to date.Identify peptides through the mass spectrometry combined with RNA sequencing.The peptidomics analysis on Bufo gargarizans ravel abundant novel toad peptides.Molecular cloning was applied following MS/MS to validate a short domain of CL4590.Skins of anurans (frogs and toads) are rich sources of bioactive peptides. However, the peptides secreted by the skin glands of Bufo gargarizans, the most common toad in China, remained unexplored to date. Here, a strategy combines LC–MS/MS, RNA sequencing and bioinformation analysis was applied to unravel the peptides in the Bufo gargarizans secretions. Data-dependent LC–MS/MS acquisitions of intact peptides followed by automated chromatographic alignment, De novo analysis, database and homology searches with manual validations showed that the venom is composed by 939 features, with masses ranging from 0.7–4kDa. These peptides derived from 85 proteins were identified using the PEAKS software with acquired MS and MS/MS spectra of Venenum Bufonis against the house-built protein database using De novo RNA sequencing, while only 23 peptides from 8 proteins were found when searching known amphibian database. Moreover, it was found that many peptides with high abundance in Venenum Bufonis derived from proteolytic processing of a larger precursor protein, named as CL4590. Molecular cloning was applied to validate a short domain of CL4590 to evident the accuracy of these obtained sequences. Although the bioactivities of peptides identified by MS/MS are unknown, the next function annotation showed that they may involve in the cell killing, immune and metabolic process, antioxidant activity and antimicrobial actions. Therefore, the peptidomics analysis on Bufo gargarizans discover abundant novel toad peptides which broaden our horizons on the secretion multiplicity and supplied an assortment of pharmacological candidates.