An innovative volatolomic approach employs the detection of biomarkers present in cerumen (earwax) to identify cattle intoxication by Stryphnodendron rotundifolium Mart., Fabaceae (popularly known as barbatimão). S. rotundifolium is a poisonous plant with the toxic compound undefined and widely distributed throughout the Brazilian territory. Cerumen samples from cattle of two local Brazilian breeds ('Curraleiro Pé-Duro' and 'Pantaneiro') were collected during an experimental intoxication protocol and analyzed using headspace (HS)/GC-MS followed by multivariate analysis (genetic algorithm for a partial least squares, cluster analysis, and classification and regression trees). A total of 106 volatile organic metabolites were identified in the cerumen samples of bovines. The intoxication by S. rotundifolium influenced the cerumen volatolomic profile of the bovines throughout the intoxication protocol. In this way, it was possible to detect biomarkers for cattle intoxication. Among the biomarkers, 2-octyldecanol and 9-tetradecen-1-ol were able to discriminate all samples between intoxicated and nonintoxicated bovines. The cattle intoxication diagnosis by S. rotundifolium was accomplished by applying the cerumen analysis using HS/GC-MS, in an easy, accurate, and noninvasive way. Thus, the proposed bioanalytical chromatography protocol is a useful tool in veterinary applications to determine this kind of intoxication. K E Y W O R D S biomarkers, classification and regression trees, cluster analysis, genetic algorithm for a partial least squares, HS/GC-MS, volatile organic metabolites 1 | INTRODUCTION Livestock farming is one of the most important economic activities around the world. According to the United States Department of Agriculture, the cattle stocks in 2019 were 1.002 billion head (USDA, 2019). Due to this enormous number of animals, there is an immediate requirement for veterinary biomedical procedures to develop new analytical methods that can ensure the welfare and health of the animals. Thus, these new analytical methods aim at avoiding economic losses due to animal deaths by disorders and intoxication, as well as the spread of diseases that can affect humans by secondary contamination due to