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The aim of this work is to offer an alternative or complementary analytical tool to the time‐consuming and expensive methods commonly used for the recognition of animal species according to their hair. The paper introduces a simple and fast way for species differentiation of animal hairs called in‐sample digestion. A total of 10 European animal species, including cat, cow, common degu, dog, fallow deer, goat, horse, sika deer, rabbit, roe deer, and 17 different breeds of dogs were examined using specific tryptic cleavage directly in hair followed by matrix‐assisted laser desorption/ionization–time of flight mass spectrometry and liquid chromatography‐electrospray ionization quadrupole time of flight. Principal component analysis was used for the subsequent mass spectrometric data evaluation. This novel approach demonstrates the ability to distinguish among individual animal species, which is supported by finding characteristic m/z values obtained by the mass spectrometry for each animal species. The approach was successfully tested on two "blind" samples. On the other hand, the attempt to distinguish among hairs of different dog breeds has not been successful due to the very similar protein composition and their amino acid sequences. [ABSTRACT FROM AUTHOR]