Simple SummaryMinerals are distributed in milk in two main forms: The diffusible (or soluble) fraction, composed of free ions and inorganic salts, and the micellar fraction, composed of mineral elements located on the surface or in the inner part of casein micelles. The ratio between diffusible and micellar minerals strongly affects milk coagulation ability. The objective of this study was to investigate the ability of mid-infrared spectroscopy to predict diffusible and micellar Ca, P, K, Mg and Na in individual milk samples of Holstein Friesian cows. Overall, the accuracy of mid-infrared prediction models was moderate for Ca, P and Mg, and low for micellar K, micellar Na and diffusible Na.Milk and dairy products are major sources of minerals in human diet. Minerals influence milk technological properties; in particular, micellar and diffusible minerals differentially influence rennet clotting time, curd firmness and curd formation rate. The aim of the present study was to investigate the ability of mid-infrared spectroscopy to predict the content of micellar and diffusible mineral fractions in bovine milk. Spectra of reference milk samples (n = 93) were collected using Milkoscan™ 7 (Foss Electric A/S, Hillerød, Denmark) and total, diffusible and micellar content of minerals were quantified using inductively coupled plasma optical emission spectrometry. Backward interval partial least squares algorithm was applied to exclude uninformative spectral regions and build prediction models for total, diffusible and micellar minerals content. Results showed that backward interval partial least squares analysis improved the predictive ability of the models for the studied traits compared with traditional partial least squares approach. Overall, the predictive ability of mid-infrared prediction models was moderate to low, with a ratio of performance to deviation in cross-validation that ranged from 1.15 for micellar K to 2.73 for total P.