In comparison of herbal medicines, less attention has been paid onto animal medicines, partially attributing to the protein-enriched property. Particularly, it is still challenging to conduct quality evaluation for the animal medicines because of the lack of a fit-for-purpose analytical tool. Herein, an attempt was made to propose a workflow allowing the quality assessment of animal medicines by LC-MS/MS, and musk that is one of the most precious traditional Chinese medicines was employed as a representative case for utility illustration. After the extraction of protein from musk with a well-defined protocol, tryptic digestion was conducted to hydrolyze proteins into peptides, and the peptide-enriched sample was subjected to nanoLC-Orbitrap MS measurement. The tandem mass spectral dataset was retrieved in Human Swiss-Prot FASTA database, and the sequences together with the sources of 733 tryptic peptides, in total, were annotated. Because of the abundant distributions, eight peptides were chosen as the analytes for quantitative measurements, and their quantitative MS parameters, such as ion transitions and collision energies, were rapid optimized in an authentic compound-free manner using online energy-resolved MS (ER-MS). On the other side, the annotated peptides were structurally consolidated via synthesizing reference peptides. When the synthetic peptides were applied for parameter optimization with the authentic compound-dependent manner, the values were almost identical with those from online ER-MS measurements. After being validated with diverse assays, the developed method was applied for the simultaneous determination of eight peptides in 28 batches of musk samples, including captive (C1-C18) and wild ones (W1-W10). Significant differences took place for the content patterns of concerned tryptic peptides between the captive and wild musk samples. Trace distributions occurred for DVDAAYMNK in most batches. Captive samples were rich of QSLEASLAETEGR, TLLDIDNTR, and EVATNSELVQSGK, whereas wild samples were able to accumulate YLGYLEQLLR. Together, the present study provided a meaningful approach for the quality evaluation of musk, as well as other peptide-enriched animal medicines, even if the absences of authentic peptides.