The present invention provides a method for selectively collecting the N-terminal peptide fragments of a protein of interest whether or not the protein of interest is modified on the N-terminus. A method for selectively collecting the N-terminal peptide fragment of a protein, comprising: a protection step (1) of protecting side chain-amino groups of amino acid residues containing side chain-amino groups of a protein of interest to obtain a protected protein protected on the side chain-amino groups; a fragmentation step (2) of cleaving the protected protein into one N-terminal peptide fragment (a) containing the N-terminus of the peptide of interest and one or more of peptide fragments (b) other than the N-terminal peptide fragment (a); and a separation step (3) of separating the N-terminal peptide fragment (a) from the other peptide fragments (b) by selectively eluting the N-terminal peptide fragment (a) based on the difference in their reactivity or affinity to substrate, wherein the selective elution is achieved either by allowing the other peptide fragments (b) to bind to the substrate while allowing the N-terminal peptide fragment (a) to elute, or by allowing the N-terminal peptide fragment (a) to bind to the substrate while allowing the other peptide fragments (b) to elute and subsequently eluting the bound N-terminal peptide fragment (a).