Background Aortic stenosis (AS) is the most common cause of valve replacement and its severity is mainly assessed by transthoracic Doppler echocardiography (TTE) to quantify valve effective orifice area (EOA) as determined by the continuity equation. In a previous study we demonstrated that EOA can be directly determined with 2D flow MRI downstream of the AS using the jet shear layer detection (JSLD) method and the acoustical source term (AST) concept. However, both TTE and 2D flow MRI rely on the measurement of local and single-directional velocities and thus incomplete assessment of the complex post-valve flow dynamics in a significant proportion of patients. 3D CINE phase contrast MRI with 3-directional velocity encoding (4D flow MRI) may improve EOA estimation by the JSLD method coupled with full volumetric coverage of ascending aortic 3D blood flow. The objective of this study was to validate 4D flow MRI based EOA estimation using an in-vitro stenosis phantom and to apply the technique for the in-vivo measurement of the valve EOA compared to the reference standard 2D flow MRI. Methods