Identification of the sex of fossil and archaeological animal remains offers many insights into their demography, mortality profiles and domestication pathways. However, due to manifold factors, sex determination of fossils is often impossible. To overcome this, we have developed an innovative protocol to determine animal’s sex applying label-free quantification (LFQ) of two unique AmelY peptides ‘LRYPYP’ (AmelY;[M+2]2+ 404.7212 m/z) and ‘LRYPYPSY’ (AmelY;[M+2]2+ 529.7689 m/z) that are solely present in males that permitted determination of the animal’s sex. We examine the application of this method to eight (8) modern cattle (Bos taurus) and (12) twelve fossil (Bos) samples from a Neolithic sites of Beisamoun in Israel (8th–7th millennium BC). Our methodology correctly determinate sex in all modern and ancient samples.