Ovarian follicle selection plays an important role in the reproduction of sexually mature hens, and this process can directly affect the growth and development of follicles until the final ovulation, thus affecting laying performance and fecundity of hens. In the laying hen ovary, one follicle from a cohort of 8-13 follicles of 6-8 mm in diameter is selected daily to enter the preovulatory hierarchy. In this study, we globally compared the proteomes of chicken ovarian follicles before and after follicle selection. A total of 5883 proteins were identified in the proteomes of chicken 6-8 mm prehierarchical follicles and 12-15 mm hierarchical follicles. 259 proteins are differentially expressed in 12-15 mm hierarchical follicle compared with prehierarchical follicle, of which 175 proteins are up-regulated and 84 proteins are down-regulated. The Gene Ontology enrichment of differentially expressed proteins revealed enriched GO terms for peptidase activity, acrosin binding for their molecular function and in the process of negative regulation of peptidase activity, and regulation of fertilization. The KEGG pathway analysis indicated that differentially expressed proteins were enriched for ribosome, lysine degradation, and endocytosis pathways. Nine differentially expressed proteins including vitellogenin-1 were validated with Parallel Reaction Monitoring (PRM) analysis, and their functions were discussed. This study provided a global proteomic view of the development of chicken ovarian follicles, which will serve as a foundation for understanding the molecular signatures and pathways of follicle selection in hens.