Peptides modified by pyridoxal-5'-phosphate (PLP), linked to a lysine residue via reductive amination, exhibit distinct spectral characteristics in the collision-induced dissociation (CID) tandem mass (MS/MS) spectra that are described here. The MS/MS spectra typically display two dominant peaks whose m/z values correspond to neutral losses of [H3PO4] (-98 Da) and the PLP moiety as [C8H10NO5P] (-231 Da) from the precursor peptide ion, respectively. Few other peaks are observed. Recognition of this distinct fragmentation behavior is imperative since determining sequences and sites of modifications relies on the formation of amide backbone cleavage products for subsequent interpretation via proteome database searching. Additionally, PLP-modified peptides exhibit suppressed precursor ionization efficiency which diminishes their detection in complex mixtures. Presented here is a protocol which describes an enrichment strategy for PLP-modified peptides combined with neutral loss screening and peptide mass fingerprinting to map the PLP-bonding site in a known PLP-dependent protein. This approach represents an efficient alternative to site-directed mutagenesis which has been the traditional method used for PLP-bonding site localization in proteins.