Identification of the sex of modern, fossil and archaeological animal remains offers many insights into their demography, mortality profiles and domestication pathways. However, due to manifold factors, sex determination of such remains is often impossible. To overcome this, we have developed an innovative protocol to determine an animal 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 tooth enamel of males. we applied this method to eight (8) modern cattle (Bos taurus) and (12) twelve archaeological samples of Bos from a Neolithic sites of Beisamoun, Israel (8th–7th millennium BC). Our methodology correctly identified the sex of the modern samples and enabled determination of sex in the archaeological samples.