Royal jelly (RJ) is a complex, creamy secretion produced by the glands of worker bees. Due to its health-promoting properties, it is used by humans as a dietary supplement. However, RJ compounds are not fully characterized yet. Hence, in this research, we aimed to broaden the knowledge of the proteomic composition of fresh RJ. Water extracts of the samples were pre-treated using combinatorial hexapeptide ligand libraries (ProteoMiner^TM kit), trypsin-digested, and analyzed by a nanoLC-MALDI-TOF/TOF MS system. To check the ProteoMiner^TM performance in the MS-based protein identification, we also examined RJ extracts that were not prepared with the ProteoMiner^TM kit. We identified a total of 86 proteins taxonomically classified to Apis spp. (bees). Among them, 74 proteins were detected in RJ extracts pre-treated with ProteoMiner^TM kit, and only 50 proteins were found in extracts non-enriched with this technique. Ten of the identified features were hypothetical proteins whose existence has been predicted, but any experimental evidence proves their in vivo expression. Additionally, we detected four uncharacterized proteins of unknown functions. The results of this research indicate that the ProteoMiner^TM strategy improves proteomic identification in complex biological samples. Broadening the knowledge of RJ composition may contribute to the development of standards and regulations, enhancing the quality of RJ, and consequently, the safety of its supplementation.