Abstract: The presence of high-abundance proteins in royal jelly makes its proteomic research very difficult. In this work to find the most suitable protein digestion method for royal jelly samples, two kinds of royal jelly from chestnut and rape flowers were used to study on proteomics using ultra-high-resolution mass spectrometry, and four protein sample pretreatment methods were used, including filter-aid sample preparation (FASP) digestion method, in-solution digestion method, fractionated in-gel protein fractionation digestion method and in-solution digestion with peptide fractionation method. The peptides were detected by liquid chromatography coupled to ultra-high-resolution mass spectrometry. Finally, under the condition of false discovery rate (FDR) less than 1%, 154, 106, 316, and 213 proteins were respectively identified in four methods from the chestnut royal jelly. 124, 121, 243, and 198 proteins were respectively identified in the four methods from the rape royal jelly. The in-gel protein fractionation digestion method can effectively separate high and low abundance proteins and identify more proteins and peptides. Meanwhile, the unfractionated FASP digestion method is a cost-efficient method, which can identify a considerable number of proteins and peptides. There is complementarity among different methods for identifying proteins. A total of 445 proteins were identified in all the experimental methods from the two kinds of royal jelly, representing the largest number of identified royal jelly proteins so far. The research results provide technical support for the study of royal jelly proteomics, and the identified new royal jelly protein species can provide a reference for the functional study of royal jelly proteins.