The lifespan of biological heart valve prostheses available in the market is limited due to structural alterations caused by calcium phosphate deposits formed from blood plasma in contact with the tissues. The objective of this work is to present a comparative methodology for the investigation of the formation of calcium phosphate deposits on bioprosthetic and tissue-engineered scaffolds in vitro and the influence of mechanical forces on tissue mineralization. Based on earlier investigations on biological mineralization at constant supersaturation, a circulatory loop simulating dynamic blood flow and physiological pressure conditions was developed. The system was appropriately adapted to evaluate the calcification potential of decellularized (DCV) and glutaraldehyde-fixed (GAV) porcine aortic valves. Results indicated that DCV calcified at higher, statistically nonsignificant, rates in comparison with GAV. This difference was attributed to the tissue surface modifications and cell debris leftovers from the decellularization process. Morphological analysis of the solids deposited after 20 h by scanning electron microscopy in combination with chemical microanalysis electron-dispersive spectroscopy identified the solid formed as octacalcium phosphate (Ca8(PO4)6H2·5H2O, OCP). OCP crystallites were preferentially deposited in high mechanical stress areas of the test tissues. Moreover, GAV tissues developed a significant transvalvular pressure gradient increase past 36 h with a calcium deposition distribution similar to the one found in explanted prostheses. In conclusion, the presented in vitro circulatory model serves as a valuable prescreening methodology for the investigation of the calcification process of bioprosthetic and tissue-engineered valves under physiological mechanical load.