Background: Previous studies indicated that the three-dimensional features of the mitral valve (MV) have a significant impact on MV disease. However, quantification of MV with manual tracing software was too time-consuming for routine clinical practice. This study was performed to investigate the efficacy and accuracy of MV quantification with a novel highly automated commercially available software package developed for this purpose. Methods: Using the manual tracing and automated package, two expert sonographers and one cardiologist individually analyzed three-dimensional datasets acquired with transesophageal echocardiography from 74 patients (15 with functional mitral regurgitation, 32 with MV prolapse, and 27 normal subjects) retrospectively. Time for analysis and inter-observer agreement were compared between the two methods, and agreement of measurements was analyzed using Cronbach's alpha. Results: Time for analysis using the automated package was significantly shorter than manual tracing (whole cohort, 260 +/- 65 vs. 381 +/- 68 seconds, P < 0.001; functional mitral regurgitation, 234 +/- 42 vs. 378 +/- 64 seconds, P < 0.001; MV prolapse, 293 +/- 69 vs. 407 +/- 67 seconds, P < 0.001; normal controls, 235 +/- 52 vs. 351 +/- 60 seconds, P < 0.001). There was good agreement among all three observers using both methods, and measurements with the automated package agreed well with the manual tracing values. Conclusions: The novel automated software package reduced time for quantification of MV with similar accuracy compared to the manual method. Automated quantification is useful and may be a key to widespread adoption of three-dimensional quantification in clinical practice.