Objectives: Reference values of hemodynamic parameters for the assessment of prosthetic heart valves are necessary and ideally need to be provided by entities independent of valve manufacturers. The aim of this in vitro study was thus to provide normal reference values of effective orifice area (EOA) for different models and sizes of mitral prosthetic valves and to assess the determinants of EOA and mean transvalvular pressure gradient (mTPG). Methods: We tested 4 models of mechanical prostheses (1 mono-leaflet and 3 bileaflet) and 4 models of bioprostheses (2 bovine pericardial and 2 porcine) on a double activation pulsed duplicator specifically designed and optimized for the assessment of the hemodynamic performance of mitral prosthetic valves. The hemodynamic conditions were standardized and included for bioprostheses: two mitral flow volumes, three mean aortic pressure, two heart rates and three E/A ratios. The EOAs were measured by Doppler-echocardiography using the same method (continuity equation) as the one used in the clinical setting. Overestimation in term of EOA was defined according with guidelines as > 0.25cm2. Results: EOA reference values were: for mono leaflet prosthesis (Medtronic Hall 7700, size 25 to 31mm): 2.29 and 3.49, for bi-leaflet prosthesis (St. Jude Medical Master and Master HP, sizes 25 to 33mm, On-X valve, sizes 27-29mm): 1.34 and 4.74 cm2; for porcine bioprostheses (Medtronic Mosaic CINCH, sizes 25 to 31mm, St. Jude Epic 100, sizes 25 to 33mm): 1.35 and 3.56 cm2; for bovine pericardial bioprosthetic valves (Edwards Perimount 6900P and Magna Ease 7300, sizes 25 to 33mm): 1.67 and 2.36 cm2. There were some discrepancies between the normal reference EOAs measured in this study versus those provided by prosthesis manufacturers or by literature. The bioprosthesis EOAs were found lower than the manufacturers' values in 32% by 0.57±0.28cm2 in average vs in 7% when compared to values presented in the literature by 0.43±0.17cm2. The relationship between EOA and the internal orifice area (IOA) varied according to the type of prosthesis. The EOA was close to the IOA in mechanical valves (regression slopes 0.87 to 0.99) but much smaller than IOA in bioprosthetic valves (slopes 0.25-0.30). The EOA was influenced by prosthesis diameters, prosthesis stent diameter and height and mTPG was influenced by EOA and heart rate. Conclusion: This study provides normal reference values of EOAs for several frequently used mitral prostheses. This information may be helpful to identify and quantify a prosthetic valve dysfunction and prosthesis patient mismatch.