A method for detecting a protein(s) that interact(s) with a target substance(s), said method comprising: a transcription step of transcribing a cDNA library encoding a candidate protein(s) to prepare an mRNA library; an assignment step of preparing an mRNA-protein assignment molecular library from said mRNA library prepared in the transcription step; a selection step of selecting an mRNA-protein assignment molecule(s) that interact(s) with a target substance(s) from the mRNA-protein assignment molecular library prepared in the assignment step; an amplification step of preparing a cDNA library encoding the candidate protein(s) by nucleic acid amplification based on the mRNA portion(s) of said mRNA-protein assignment molecule(s) selected in the selection step; and repeating Steps to using said cDNA library prepared in the amplification step; wherein said method comprises: (a) preparing, in each of a plurality of times of preparation of a cDNA library among the initial preparation of a cDNA library and the round(s) of preparation of a cDNA library in the later amplification step(s), the cDNA library using a primer(s) having a sequence(s) specific to the time of preparation; (b) mixing the cDNA libraries prepared using said primers having sequences specific to the times of preparation, and determining sequences in the cDNA library mixture; (c) subjecting the determined sequences to measurement of the number(s) of molecules encoding the same candidate protein(s) for each time of preparation based on the sequence(s) specific to the time of preparation; and (d) detecting, as the protein(s) that interact(s) with said target substance(s), a candidate protein(s) encoded by a molecule(s) that significantly increase(s) as the preparation rounds proceed. a transcription step of transcribing a cDNA library encoding a candidate protein(s) to prepare an mRNA library; an assignment step of preparing an mRNA-protein assignment molecular library from said mRNA library prepared in the transcription step; a selection step of selecting an mRNA-protein assignment molecule(s) that interact(s) with a target substance(s) from the mRNA-protein assignment molecular library prepared in the assignment step; an amplification step of preparing a cDNA library encoding the candidate protein(s) by nucleic acid amplification based on the mRNA portion(s) of said mRNA-protein assignment molecule(s) selected in the selection step; and repeating Steps to using said cDNA library prepared in the amplification step; (a) preparing, in each of a plurality of times of preparation of a cDNA library among the initial preparation of a cDNA library and the round(s) of preparation of a cDNA library in the later amplification step(s), the cDNA library using a primer(s) having a sequence(s) specific to the time of preparation; (b) mixing the cDNA libraries prepared using said primers having sequences specific to the times of preparation, and determining sequences in the cDNA library mixture; (c) subjecting the determined sequences to measurement of the number(s) of molecules encoding the same candidate protein(s) for each time of preparation based on the sequence(s) specific to the time of preparation; and (d) detecting, as the protein(s) that interact(s) with said target substance(s), a candidate protein(s) encoded by a molecule(s) that significantly increase(s) as the preparation rounds proceed.