Tooth enamel is the hardest, densest and most mineralized tissue in vertebrates. This is due to the high crystallinity of enamel. During enamel formation, proteins responsible for mineralization are degraded by proteases, which results in mature enamel having less than 1% proteinaceous material, mostly as peptides. Many toxicological studies have taken advantage of the stability of tooth enamel to study heavy metal exposure, however few studies have been successful in identifying peptides from the enamel, especially from a single tooth. Furthermore, amelogenin, the most abundant protein involved in tooth development, is expressed from both the X and Y chromosomes and is dimorphic. Sequencing of the gender dimorphic peptide regions may be useful in determining gender, especially when no other biomaterial is available nor intact DNA remains. In light of this, a method employing nanoflow liquid chromatography (nanoLC) electrospray ionization tandem mass spectrometry (MS/MS) was used to analyse peptides released through an acid etch of the enamel from individual teeth. Two approaches were investigated, one with trypsin digest following acid etch and one without. Peptide identification was accomplished using typical proteomics methodology by searching against the human proteome. Peptides from the major enamel structural proteins were identified including; amelogenin isoforms, ameloblastin, and enamelin. Furthermore, Y chromosome-specific amelogenin peptides were also detected in mature enamel. Peptides were identified from the enamel of single teeth on present-day and archaeological samples in a non-destructive and minimally invasive method by nanoLC-MS/MS. The identification of tooth enamel specific peptides with this approach allows for its potential applications in forensic analysis and archaeological studies.