Development and progression of myxomatous mitral valve disease (MMVD) in domestic dogs is unpredictable and pathobiology still unclear. The American College of Veterinary Internal Medicine (ACVIM) perceived that mayor improvement in management of diseased dogs would be timely diagnosis, especially detection of transition from MMVD stage B1 into B2. Thus, in this study we compared by tandem mass tag (TMT) protocol and mass spectrometry (MS) acquired quantitative proteome profiles of serum collected from healthy (control) (N=12) and dogs diagnosed with different stages of naturally occurring MMVD: B1 (N=13), B2 (N=12) and C (N=13). Prior to proteomic analysis dogs were distinguished into experimental categories based on echocardiography results. Serum biochemistry and concentrations of three cardiac biomarkers (galectin-3, suppression of tumorigenicity 2 and asymmetric dimethylarginine) were performed to obtain better characterization of healthy/control group and MMVD cases.