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• A fiber-optic spatially-resolved spectroscopy (SRS) setup measured milk NIR spectra. • The setup evaluated 30 different illumination-to-detection distances (1.1–4 mm) • Shorter illumination-to-detection distances were optimal for lactose prediction. • Larger illumination-to-detection distances were optimal for fat and protein. • The accuracy of the composition predictions are well within the ICAR requirements. To support in-line quality control of raw milk and ensure the close monitoring of the metabolic and udder health of dairy cows, we propose a fiber-optic spatially-resolved spectroscopy (SRS) setup. This setup allows to vary the interaction of long-wave near-infrared (LW-NIR) light with the milk by submerging a separate optical illumination and detection fiber into the sample and altering their relative distance to optimize measurements for specific milk component(s). We evaluated this approach for predicting milk fat, protein, and lactose content and determined the optimal illumination-to-detection distances for each milk component. The region between 1.1 and 1.8 mm was optimal for lactose, and between 2.2 and 3.8 mm for fat and protein. These distance ranges resulted in a root-mean-square error of prediction (RMSEP) of less than 0.10 % weight/weight (wt/wt) for milk fat, and lower than 0.13 % (wt/wt) for protein and lactose. Integration of these distances into a fiber-optic SRS reflectance probe would allow to simultaneously determine the fat, protein, and lactose content of raw milk in-line with high accuracy. [ABSTRACT FROM AUTHOR]