It is challenging to achieve in-depth quality analysis for animal-originated medicinal materials (AMMs) owing to containing abundant metabolites and proteins. RPLC-MS/MS intrinsically bears the ability to simultaneously monitor metabolites and peptides. Hence, its potential towards merging quasi-quantitative metabolomics and tryptic proteomics characterization is therefore assessed in current study, and a well-known AMM namely Bufonis Venenum (BV, Chinese name: Chansu) was employed as a proof-of-concept. Qualitative information of metabolites was acquired by RPLC-Qtof-MS and translated to plausible structures through careful "spectrum-to-structure" analysis. Bottom-up proteomics was conducted to characterize the tryptic peptidome using nanoLC-QExactive HF orbitrap-MS. Quantitative MS/MS parameters of either metabolites (72 ones) or tryptic peptides (28 unique peptides) were optimized using online energy-resolved MS and applied to configure RPLC-selected-reaction monitoring (RPLC-SRM) program. Ultimately, SRM response of each analyte was converted to quasi-content by serially diluting a so-called universal metabolome standard (UMS) solution and building regressive calibration curve set to achieve widely quasi-quantitative metabolomics and proteomics. Although being sourced from identical species, significant differences occurred among the metabolite and protein profiles between BV and toad skins, and bufadienolides (i.e., 3-(N-adipoyl-argininyl)-gamabufotalin/isomer) along with several tryptic peptides (i.e., ISGLIYEETR sourced from Histone H4) served as the primary differential variables. Above all, RPLC-SRM is a promising analytical tool for in-depth quality evaluation of AMMs, and more importantly, the workflow described here is a fit-for-purpose pipeline to merge quasi-quantitative metabolomics and bottom-up proteomics.Copyright © 2022 Elsevier B.V. All rights reserved.