PURPOSE: Isolate renewable and cost-efficient affinity reagents that will facilitate the detection of p24, the capsid protein of Human Immunodeficiency Virus (HIV), by screening phage-displayed combinatorial peptide libraries and identifying peptide ligands. METHOD: Four in-house combinatorial peptide libraries were screened for binders in three progressive rounds against monomeric p24 protein. Peptide binders were characterized by ELISA and Surface Plasmon Resonance (SPR) and one peptide sequence was evaluated in a lateral flow assay (LFA). RESULT: We identified 26 unique peptide sequences that exhibit varying phage ELISA signals above background for p24. We subsequently validated the binding of one linear and two cyclized peptide sequences with synthetic peptides. Alanine-scanning identified several residues critical to binding in the linear peptide. The linear peptide could be used for p24 detection in ELISA and LFAs. CONCLUSION: Phage-displayed combinatorial peptide libraries are suitable for isolation of binders against p24 and potentially other targets. Upon identification of a minimal binding sequence, the subsequent characterization and future optimization of it can lead to a variety of diagnostic assays.