An in vitro technique has been developed at Edinburgh University that uses renneted milk as a blood analogue fluid for assessing the flow-induced clotting of heart valves. There were two main objectives with the current research. The first aim was to further confirm the milk technique’s ability to simulate thrombus formation, and the second aim was to assess the impact of test conditions on in vitro clotting. In support of the first aim, confocal laser scanning microscopy (CLSM) was utilised to observe milk coagulation in a stagnation point flow chamber at different shear rates, for comparison with platelet deposition. Comparison of the coagulation of milk with that of whole blood was achieved by testing twelve different heart valves, representing all available valve types, in the existing heart chamber and comparing localised milk clotting with sites of reported thrombus formation. Valves were then retested in a new heart chamber, which consisted of a flexible model of the left ventricle and a straight walled aortic section, or one incorporating the sinuses of Valsalva, mounted on top. Deposition of milk in the stagnation point flow chamber was dependent on shear stress; there was a minimum shear stress for protein alignment, and a maximum shear stress above which proteins did not deposit. This compared favourably with reports of limiting shear stresses for the growth of platelet aggregates, which have also been shown to align with flow in the presence of sufficient shear forces. The milk test successfully generated clots in specific, reproducible locations on all of the rigid valves tested, and this clotting compared favourably with reported sites of thrombus formation in vivo. However, localised milk clot did not form on a flexible trileaflet valve prototype; it was suspected that this may have been due to the valve not functioning properly under the current test conditions. Clot development was affected by both chamber design and exposure time to rennet, the latter being significant for all valve types. Localised clotting was not observed if the valve was exposed to renneted milk in the primary stage of coagulation. Clot formation on the caged-ball valves and the monoleaflet valves appeared to be independent of the test chamber shape, but clot formation on the bileaflet valves was not; interaction of the downstream flow with the chamber wall appeared to govern clot development.