Rationale: Sex estimation by analysis of amelogenin peptides in archaeological and fossil material has recently been gaining great traction within the fields of archaeology and palaeontology. Current widely used proteomic amelogenin sex estimation methods are hindered by relatively long mass spectrometric run times, or targeting peptides specific to human amelogenin proteins. Untargeted, high-throughput amelogenin sexing would be invaluable for a range of applications, from sex estimation of remains at mass grave sites to broadening the application of rapid amelogenin sexing to non-hominin species for husbandry and evolutionary studies. Methods: A new acid etch amelogenin analysis protocol followed by Evosep-LC-TIMS-TOF mass spectrometry is presented, providing global peptide data through rapid mass spectrometric methods in under 20 minutes per sample (including sample preparation, mass spectrometric acquisition and data processing). Furthermore, this method is applied to a preliminary study of both modern and archaeological Bos taurus remains, alongside archaeological human remains, showing the potential of straightforward application of this rapid amelogenin sexing method to a range of taxa. Results: Application of the developed Evosep-LC-TIMS-TOF mass spectrometry shows the novel acid etch approach improved peptide counts. Furthermore, rapid untargeted mass spectrometry using the Evosep-LC-TIMS-TOF gave comparable peptide counts to conventional long untargeted methods, while maintaining similar, or faster, acquisition times to those reported in methods targeting specific human amelogenin peptides. Conclusions: Rapid, untargeted Evosep-LC-TIMS-TOF mass spectrometry was successfully implemented in sex estimation of modern and archaeological material from Bos taurus and human teeth. This demonstrates an advancement in low-cost, high-throughput amelogenin sex estimation, for both human and zooarchaeological applications.