Mechanical heart valves carry the disadvantage of lifelong antithrombotic therapy, due to the high risk of thrombus formation on the valve surface. Current diagnostic methods are incapable of detecting thrombus formation in an early stage. This article investigates a new diagnostic method, based on the analysis of the acoustic signal produced by the valve. This method should be capable of early detection of malfunction, thus permitting targeted medication and reducing valve-related complications and mortality. A measurement setup assuring optimal signal quality was developed, and a signal analysis program was implemented and validated on an in vitro mock circulatory loop. Next, four sheep were implanted with a bileaflet mechanical valve. The signals of their valves developing thrombosis were assessed on a weekly basis before explantation. Three sheep were sacrificed shortly after detection of malfunction according to the newly developed method. In each case, thrombus or membrane formation was detected on the leaflets upon explantation. In one sheep, no malfunction was found in the analysis, which was also confirmed by the condition of the valve upon explantation. These preliminary results indicate that acoustical analysis of mechanical heart valves permits early detection of valvular malfunction. Further research with more in vitro and animal testing is required to statistically validate these findings.