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To discover novel biomarkers of hypopharyngeal gland (HG) development in honeybees, a proteomic method combining the isobaric tag for relative and absolute quantification (iTRAQ) labeling technique with liquid chromatography-electrospray ionization mass-spectrometry (LC-ESI-MS/MS) in a TripleTOF 5600 instntment was employed to identify differentially expressed proteins (DEPs) during HG development.A total of 1282 proteins were identified from five samples of worker bees, 320 of which were differentially expressed.Comparison of samples at day 6, 9, 12, and 16 of development relative to day 3 led to the unambiguous identification of 112, 117, 127, and 127 DEPs, respectively.Forty-three of the DEPs were identified for the first time in our dataset.DEPs were grouped into the following functional categories: binding, catalytic activity, cell & cell part, metabolic process, cellular process, organelle part, macromolecular complex, structural molecular organization, developmental process, and biological regulation.More than 10 key high-abundance proteins were involved in signaling pathways related to ribosome function and protein processing in the endoplasmic reticulum.Our approach demonstrates that a dynamic set of differentially expressed proteins regulate the protein network that supports royal jelly (RJl) initiation, fastigium, and degradation during HG development.These results should enhance our understanding of the molecular basis of HG development and RJ regulation.