Biological sex is a key information for archaeological and forensic studies, which can be assessed in enamel using proteomics. However, the lack of a standard approach for fast and accurate sex identification limits the reach of proteomics applications. Here, we introduce a targeted proteomics-based workflow that builds on a rapid, non-destructive, sensitive, and quantitative mass spectrometry (MS) strategy for the determination of biological sex from human dental enamel, including a R-Shiny interface for the data analysis. The method was first developed and optimised using modern teeth material and then validated on an independent set of modern deciduous teeth of known sex. The assay was then applied to archaeological material and demonstrated unprecedented specificity and scalability for molecular sex inference, with the possibility of analysing > 300 individuals in less than two days of MS measurement time, and with subsequent confident sex assignment in less than a day of data analysis.