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Interest in the fatty acid composition of dairy products is increasing; however, the measurement of fatty acids requires using gas-liquid chromatography. Although this method is suitable, it involves a time-consuming procedure, expensive reagents, and qualified staff. By comparison, the mid-infrared (MIR) spectrometry method could be a good alternative for assessing the fatty acid profile of dairy products. The objective of this study was to explore the calibration of MIR spectrometry for estimating fatty acid concentrations in milk and milk fat. Estimated concentrations in milk fat were less reliable than those for the same fatty acids in milk. Results also showed that when the fatty acid concentrations in milk increased, the efficiency of the infrared analysis method in predicting these values simultaneously increased. Selected prediction equations must have a high cross-validation coefficient of determination, a high ratio of …