Animal welfare issues are detected on-farm using animal-based indicators such as body injuries requiring a visit by a trained assessor. The overall goal of this study is to develop precision markers that could be routinely recorded to detect cows experiencing lower level of welfare. A specific objective is to demonstrate that Fourier transform infrared (FTIR) spectroscopy can detect changes in milk composition related to cows subjected to housing treatments whose level of welfare is characterized by a given level of movement restriction at the stall. A total of 48 cows were assigned to 4 tie-rail configurations. Tie-rail heights were 122, 122, 112 and 102 cm and forward positions were 18, 36, 18 and 36 cm for treatments T1, T2, T3 and T4, respectively. Cows were assigned to 6 blocks to account for parity, days in milk and barn location effects. The trial lasted 10 weeks. Milk mid-infrared spectra were collected on weekly basis. Different preprocess treatments were applied and regions 3061–2803 cm⁻¹, 1797–1681 cm⁻¹ and 1612–925 cm⁻¹ were retained for analysis. Long-term average spectra were calculated for each cow using spectra collected in wk 8–10 of treatment. Principal component analysis was applied to spectral averages and scores of principal components (PCs) were tested for treatment effect by multivariate ANOVA and mixed modeling. Normalized first derivative long-term averaged spectra revealed a significant treatment effect on PC7 (P = 0.01). Tukey’s means multiple comparisons of treatment scores for PC7 revealed that T3 was significantly different from T1 (P = 0.02), T2 (P = 0.04) and T4 (P = 0.04). Loading spectrum of PC7 revealed intense peaks that could be assigned to biomarkers related to negative energy balance, such as citrate and acetone. This observation suggests that T3 might have been restrictive for cows to access feed. T3 was the only treatment resulting in an increase in neck injuries on 2 locations of the cows’ neck corroborating findings from spectral analysis. Milk FTIR spectroscopy showed promising results in detecting welfare status in dairy cows.