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Background: Ticks and tick-borne diseases pose significant challenges to cattle production, thus the species identification of ticks and knowledge on their presence, abundance, and dispersal are necessary for the development of effective control measures. The standard method of inspection for the presence of ticks is the visual and physical examination of restrained animals, but the limitations of human sight and touch can allow larval, nymphal, and unfed adult ticks to remain undetected due to their small size and site of attachment. However, Raman spectroscopy, an analytical tool widely used in agriculture and other sectors, shows promise for the identification of tick species in infested cattle. Raman spectroscopy is a non-invasive and efficient method that employs the interaction between molecules and light for the identification of the molecular constituents of specimens. Methods: Raman spectroscopy was employed to analyze the structure and composition of tick feces deposited on host skin and hair during blood-feeding. Feces of 12 species from a total of five genera and one subgenus of ixodid ticks were examined. Spectral data were subjected to partial least squares discriminant analysis, a machine-learning model. We also used Raman spectroscopy and the same analytical procedures to compare and evaluate feces of the horn fly Haematobia irritans after it fed on cattle. Results: Five genera and one sub-genus at overall true prediction rates ranging from 92.3 to 100% were identified from the Raman spectroscopy data of the tick feces. At the species level, Dermacentor albipictus, Dermacentor andersoni and Dermacentor variabilis at overall true prediction rates of 100, 99.3 and 100%, respectively, were identified. There were distinct differences between horn fly and tick feces with respect to blood and guanine vibrational frequencies. The overall true prediction rate for the separation of tick and horn fly feces was 98%. Conclusions: Our findings highlight the utility of Raman spectroscopy for the reliable identification of tick species from their feces, and its potential application for the identification of ticks from infested cattle in the field. [ABSTRACT FROM AUTHOR]