UNLABELLED: Mitral regurgitation (MR) is a common valvular lesion frequently caused by mitral valve prolapse (MVP). Surgical intervention in MVP patients with significant MR is predicated on symptoms and measures of left ventricular dysfunction. Because these indicators may be subjective or imprecise, serological biomarkers of disease could be a valuable adjunct to standard evaluation. This study aimed to identify such biomarkers by a proteomics approach. Two pooled plasma samples from 24 MVP subjects with MR (MVP/MR) and 24 non-MVP individuals were treated with the combinatorial peptide ligand library (CPLL) beads prior to iTRAQ labeling and ESI-MS/MS. Lower levels of haptoglobin, platelet basic protein (PBP), and complement component C4b were observed in the MVP/MR as compared to the control sample. These findings were verified by ELISA testing of each of the 24 paired samples, and another 42 matched cases and controls. The AUC values, sensitivities and specificities for (i) haptoglobin, (ii) PBP, (iii) C4b, and (iv) all 3 proteins in combination were (i) 0.813, 76%, 74%; (ii) 0.721, 56%, 77%; (iii) 0.689, 83%, 49%; and (iv) 0.840, 89%, 67%, respectively. In conclusion, haptoglobin, PBP, and C4b are down-regulated in MVP/MR. Their value as serological biomarkers of valvular pathology should be further explored. BIOLOGICAL SIGNIFICANCE: We report the first study that performed comparative proteomics of clinical human plasma samples to identify novel diagnostic biomarkers for mitral valve prolapse (MVP) patients with moderate to severe mitral regurgitation (MR). MR is a common valvular lesion that can be complicated by heart failure, sudden death and atrial fibrillation, yet many patients with severe MR are asymptomatic. Our results revealed reduced levels of haptoglobin, platelet basic protein (PBP), and complement component C4b in the MVP/MR patients as compared to the matched control cases. The plasma proteomics findings were subsequently confirmed by ELISA. Each of these candidate biomarkers has a putative role in the pathophysiology of MVP/MR, further supporting their roles in detection and possibly surveillance and prognostication of this disease.