We sought to build a robust pulsatile flow model permitting comparison of 3D Color Doppler and phase contrast magnetic resonance (PCMR) flow and orifice area measurements of prosthetic valve function that would provide an in vitro gold standard for evaluation of the accuracy of new imaging techniques. A multi-modality compatible, pulsatile linear actuation system, control software, and mock circulation loop was designed and created using non-ferromagnetic components, NI Labview, and rapid prototyping. The developed system’s ability to create pulsatile, controlled flow was assessed by examining peak diastolic flow, diastolic period, and diastolic flow volume (DFV) of 12 consecutive cardiac cycles at 3 different DFVs. Reproducibility of flow was tested by examining the systems ability to generate the same target DFV after draining and re-configuring the flow loop components. Furthermore, the system was evaluated by CMR and echocardiography to examine image quality, DFV quantification, and an initial functional assessment of prosthetic mitral valve (MV) orifice area by four different imaging methods. The system was able to reproducibly create prescribed flow volumes across 31 mm bioprosthetic and mechanical MV’s under a range of flow conditions. In vitro image quality for the identification of MV sewing ring, struts and leaflets was similar to clinical imaging quality for both echocardiographic and MRI methods. For the quantification of DFV, the PCMR method demonstrated an error range of 1.4–9.2% and a mean difference of 4 ± 2 mL/beat compared to the flowmeter reference standard. Across the three flow conditions the bioprosthetic MV diastolic flow area was statistically similar by MRI planimetry, phase contrast MRI planimetry, and 3D color Doppler planimetry methods (mean area 3.2 ± 0.5 cm2, ANOVA, p = NS), but the effective orifice area by 2D spectral Doppler (continuity) method was smaller (mean area 2.0 ± 0.3 cm2, ANOVA, p < 0.001). We describe the development and initial functional testing of a novel MRI-compatible flow loop system for comparison of multimodality imaging techniques to assess prosthetic valve function.