A challenge for shotgun proteomics is the identification of low abundance proteins, which is always hampered owing to the extreme complexity of protein digests and highly dynamic concentration range of proteins. To reduce the complexity of the peptide mixture, we developed a novel method to selectively enrich N-terminal proline peptides via hydrazide chemistry. This method consisted of ortho-phthalaldehyde (OPA) blocking of primary amines in peptides, reductive glutaraldehydation of N-terminal proline and solid phase hydrazide chemistry enrichment of aldehyde-modified N-terminal proline peptide.