Background: Phase contrast cardiac magnetic resonance imaging allows for the simultaneous acquisition of morphology in 3D (x, y, and z directions) and time-resolved blood flow velocities (known as 4D flow MRI). The clinical applications of this methodology continue to be developed. For this study, we sought to evaluate the methodology and accuracy of 4D flow MRI for the quantification of diastolic flow volume across bioprosthetic mitral valves (BMV) in a controlled and reproducible in vitro system. Methods: BMV of 3 sizes (27, 29, and 31 mm) were consecutively mounted in an MRI compatible flow loop. Flow conditions within the flow loop were set using control software that programmed a pump to generate pulsatile, physiologic ventricular ejection and filling characteristics. Diastolic flow volumes of ~70, 90, and 110 ml/beat were tested for each valve at a beat rate of 70 bpm. An in-series ultrasonic flow transducer (UFT) was used to measure flow (L/min) by which diastolic flow volumes were determined. The acquisition of 3D cine (4D flow) phase contrast velocity data was acquired in a 1.5 Tesla MRI scanner (Siemens) and flow measurements were determined “offline” at 3 locations within the valve; valve base, leaflet tips, and midway between the base and the tips. Results: When 4D flow volume measurements were compared to UFT diastolic flow volumes, the average difference was -0.2 ± 3.0 ml/beat. For all flow condition tested (n=8), the 4D flow measurements were strongly correlated to the reference standard flow meter, regardless of measurement location within the bioprosthesis (Pearson correlation, r=0.98, 0.99, and 0.99 for the base, mid, and tips location, respectively. P<0.001). Conclusion: 4D flow MRI can be used to directly assess bioprosthetic mitral valve diastolic inflow volume, with considerable accuracy compared to reference standard measure. The imaging plane chosen for trans-valvular flow analysis did not affect the accuracy of the 4D flow measurements.