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Rationale: Trade in rhinoceros horn is regulated or banned internationally in recognition of its impact on wild populations worldwide. Enforcement of the laws and regulations depends on successfully identifying when violations occur, which is complicated by the presence of alternative/imitation rhinoceros horn keratin (e.g., bovid horn keratin). In this study, we assess the potential for Direct Analysis in Real Time (DART) ionization paired with Time‐Of‐Flight Mass Spectrometry (DART‐TOFMS) to classify different keratin types from four taxonomic groups: rhinoceros, bovid, domestic horse, and pangolin. Methods: The spectra of 156 keratin samples from all five rhinoceros species (horn keratin), eight genera of bovids (horn keratin), domestic horses (hoof keratin), and all extant species of pangolins (scale keratin) were collected. Fisher ratio analysis identified the most important ions that characterized each class and these ions were used for the training model, which consisted of 143 spectra. Kernel Discriminant Analysis (KDA) was used to classify the different groups. Results: The spectra collected for each taxonomic group are distinctive. The chemotypes demonstrate that the spectra of rhinoceros, bovids, and domestic horse are similar to each other, whereas the chemotypes of pangolins show a different chemical profile. The model built by KDA resolved each taxonomic group: 95% of samples were correctly assigned using leave‐one‐out cross validation. The 13 blind samples not used in model development were all correctly classified to taxonomic source. Conclusions: DART‐TOFMS appears to be a reliable approach for taxonomic identification of keratin. This analysis can be carried out with a small sliver of keratin, with minimal sample preparation, inexpensively and quickly, making it a potential valuable tool for identification of rhinoceros horn and other keratin types. [ABSTRACT FROM AUTHOR]