This study aimed to validate a dedicated software for quantitative videodensitometric angiographic assessment of mitral regurgitation (QMR).Quantitative videodensitometric aortography of aortic regurgitation using the time-density principle is a well-documented technique, but the angiographic assessment of mitral regurgitation (MR) remains at best semi-quantitative and operator dependent.Fourteen sheep underwent surgical mitral valve replacement using 2 different prostheses. Pre-sacrifice left ventriculograms were used to assess MR fraction (MRF) using QMR and MR volume (MRV). In an independent core lab, the CAAS QMR 0.1 was used for QMR analysis. In vitro MRF and MRV were assessed in a mock circulation at a comparable cardiac output to the in vivo one by thermodilution. The correlations and agreements of in vitro and in vivo MRF, MRV, and interobserver reproducibility for QMR analysis were assessed using the averaged cardiac cycles (CCs).In vivo derived MRF by QMR strongly correlated with in vitro derived MRF, regardless of the number of the CCs analyzed (best correlation: 3 CCs y = 0.446 + 0.994x; R = 0.784; p =0.002). The mean absolute difference between in vitro derived MRF and in vivo derived MRF from 3 CCs was 0.01 ± 4.2% on Bland-Altman analysis. In vitro MRV and in vivo MRV from 3 CCs were very strongly correlated (y = 0.196 + 1.255x; R = 0.839; p < 0.001). The mean absolute difference between in vitro MRV and in vivo MRV from 3 CCs was -1.4 ± 1.9 ml. There were very strong correlations of in vivo MRF between 2 independent analysts, regardless of the number of the CCs.In vivo MRF using the novel software is feasible, accurate, and highly reproducible. These promising results have led us to initiate the first human feasibility study comprising patients undergoing percutaneous mitral valve edge-to-edge repair.