Eupolyphaga sinensis Walker is a traditional Chinese medicinal insect. However, the peptide composition differences between male and female ES, which may underlie their distinct medicinal efficacies, remain largely unexplored. In this study, we performed a quantitative peptidomics analysis using stable isotope dimethyl labeling combined with high-resolution nanoLC-MS/MS to systematically compare the peptide profiles of male and female ES. Tryptic peptides from female sources were labeled with light reagents (HCHO/NaBH3CN), while those from male sources were labeled with medium reagents (DCDO/NaBH3CN). Equal amounts of labeled samples were mixed and analyzed by Q Exactive Plus mass spectrometer. The quantitative ratios of male to female peptides were calculated based on the peak area intensities of the isotope-labeled precursor ions.