Characterization of proteomes aims to comprehensively characterize proteins in cells or tissues via two main strategies: (1) bottom-up strategy based on the separation and identification of enzymatic peptides; (2) top-down strategy based on the separation and identification of intact proteins. However, it is challenged by the high complexity of proteomes. Consequently, the improvements in peptide and protein separation technologies for simplifying the sample should be critical. In this feature article, separation columns for peptide and protein separation were introduced, and peptide separation technologies for bottom-up proteomic analysis as well as protein separation technologies for top-down proteomic analysis were summarized. The achievement, recent development, limitation and future trends are discussed. Besides, the outlook on challenges and future directions of chromatographic separation in the field of proteomics was also presented.