Being a part of cultural heritage, skeletal human remains and grave objects are often the only evidence of people who lived many years, decades, or even centuries ago. Studies of skeletons from archaeological excavations of burial grounds are kept in the collections of museums and university workplaces. Their research requires an approach that preserves them for future generations, rather than damaging them. The first task in analyzing skeletal material is to determine the biological profile of the individual, especially sex estimation. For biological sex estimation from skeletal remains, it is important to use the least destructive methods, while providing reliability. Proteomics is a method that has been developing in recent years, where the presence of two types of amelogenin protein in tooth enamel can provide reliable estimation of biological sex in recent as well as past populations. Validation of proteomic sex estimation was performed in a set of 40 teeth of a recent and sub-recent population of known sex and achieved 100% accuracy. The application concerns 15 teeth of a medieval population where sex estimation by different morphological methods gave conflicting results. Furthermore, we used the proteomics method for a set of teeth of juvenile individuals of the medieval population where gender estimation by morphological methods is not possible. Proteomic sex estimation is usually characterized as non-destructive and convenient to use. This is the first study evaluating the destructiveness of this method in a sample of 20 teeth of the first set. After chemical treatment with 3% H2O2 and 5% HCl, microscopic morphological changes on the tooth surface were detected using a scanning electron microscope (SEM), while enamel loss of approximately 10% and dentin loss of only 2% were detected using microcomputed tomography.