So far, high-efficiency and high-throughput analysis of C-terminal peptides is still full of challenges. In order to make the chemical derivatization-based strategies more practical and efficient, in this study we developed a new method, named 2-pyridinecarboxaldehyde (2PCA) and biotin labeling based C-terminomics (PBC), for high-throughput and highly efficient enrichment of C-terminal peptides.