Significance The ability to assign biological sex to human skeletal remains is a fundamental requirement in archaeology, paleoanthropology, and medico-legal sciences. While DNA sequencing can be used, it is expensive, time-consuming, and often fails due to the poor quality of the remaining DNA. An easier, more reliable, and consistently applicable method is needed. We present a method for sex determination of human remains using peptides retrieved from tooth enamel. Amelogenin is an enamel-forming protein encoded for by both chromosomes X and Y, with slight differences in their amino acid sequences. Peptides with these differences were identified by nanoflow liquid chromatography mass spectrometry and found to correctly assign sex to archaeological human remains of various chronological ages, from hundreds to thousands of years old.