Differentiating between the genus Bos and Bison from fossil bones and teeth can be difficult and even impossible due to taphonomic deterioration, which can also muddle the identification of the sex of the animal. Both are key issues for palaeobiological and palaeoecological studies, as the two species are generally considered having quite similar spatial distributions but distinct ecological preferences, and likely similar social behaviours with males separated from female groups during most of the year. However, identifications are usually limited to “Bos/Bison” and “Sex indeterminate”, which severely limits interpretations. Here, we propose a low-invasive mass spectrometry method for simultaneous taxonomic and sex determination of Bos and Bison teeth, with successful application on a Middle Pleistocene large bovid teeth from Lazaret cave. The peptides and proteins present in the enamel were directly extracted at the surface of the teeth. The proteins were then digested with trypsin or chymotrypsin and analysed by LC-MS/MS. The results obtained show that the enamelin, COL1A3 and alpha-2-glycoprotein allow for taxonomic differentiation between Bos and Bison, while AMELX/Y sequences of the same samples allow for sex identification. Both were successfully performed on the 160-120 ky archaeological teeth, without affecting the specimen. This study highlights the potential of proteomics for simultaneous taxonomic and sex determination for others fossil samples, including rare or precious material, using low-invasive high-resolution mass spectrometry. It opens up unprecedent avenues for palaeobiological studies as well as cultural and natural heritage, and will widely participate to strengthen our knowledge of past animal and human communities. Such multi-proxy approach, shall be now developed for other mammalian species.