Our cohort consisted of 15 horses with osteoarthritis (OA) and 48 without clinical signs of the disease, which were used as controls. Attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy was used to analyse serum samples (50 μL) collected from the donors. Spectral processing and multivariate analysis revealed differences and similarities, allowing for detection of biomarkers discriminating between the two cohorts. A supervised classification algorithm, namely principal component analysis coupled with quadratic discriminant analysis (PCA-QDA), was also applied to evaluate the diagnostic accuracy.