Objectives: To identify serum biomarkers differentiating dogs with and without osteoarthritis (OA). Methods: Following institutional ethical approval, 24 dogs (27.2-48.5 kg, 3.6-13.6 years) previously diagnosed with OA affecting different joints with varying severity were included in the study. Sixteen dogs (16.2-36 kg, 2.0-6.7 years) without detectable orthopaedic pathology were included as clinically healthy controls. Other disorders were ruled out by thorough clinical examinations and standard hematological and biochemical analyses. Serum was stored at -80oC until analysis of five biomarkers using commercially available enzyme-linked immunosorbent assays: Hyaloronic acid (HA), Matrix Metalloproteinase 13 (MMP-13), Procollagen Type IIA (PIIANP), Cartilage oligomeric matrix protein (COMP), and Collagen Type-2 Cleavage (C2C). Medians and range of concentrations were calculated and differences between groups were tested for significance using Mann Whitney tests (p<0.05). Results: Higher concentrations of C2C and MMP-13 were observed in dogs with OA (median, [95% CIs] C2C: 23.7 ng/ml [18.2; 45.2 ng/ml], MMP-13: 0.70 ng/ml [0.51;1.09 ng/ml]) compared to clinically healthy dogs (C2C: 12.3 ng/ml [10.1; 35.3 ng/ml], MMP 13: 0.34 ng/ml [0.27; 0.88 ng/ml]), whereas lower concentrations of PIIANP were observed in dogs with OA (11.6 ng/ml [10.7; 18.02 ng/ml] compared to clinically healthy dogs (15.8 ng/ml [11.7; 31.7 ng/ml]). Observed differences were not significant. Statement: None of the 5 biomarkers measured in the present study were useful in differentiating heterologous groups of dogs with and without OA. Further studies are, however, recommended in order to investigate possible diagnostic potentials of C2C, MMP-13 or PIIANP in some particular group of dogs with OA.