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