Tissue engineering is currently being actively investigated to ascertain if it can offer an alternative to prosthetic aortic heart valves that may overcome the current limitations of prosthetic aortic heart valves while at the same time conferring the advantages of a living autologous structure, such as biocompatibility, the capacity to grow, repair, and remodel. In vitro studies have shown tissue-engineered heart valves to have adequate structural and functional properties, indicating a promising future for heart valve tissue engineering. However, criteria are required to be able to evaluate autologous heart valves and to deem them satisfactory for clinical use. Preclinical animal studies are needed, as a precursor to long-term in vivo follow-up studies, to establish such criteria. The first challenge is to find appropriate techniques to evaluate the functionality of tissue-engineered heart valves in vivo without having to kill the animal. As such, the development of such noninvasive techniques that are able to assess the functionality of tissue-engineered heart valves is the next step in translational research. This review discusses methods of evaluating the functionality of autologous heart valves when translating from in vitro to in vivo studies and determines potential assessment criteria imperative to achieve clinical applicability of tissue-engineered heart valves in aortic valve replacement.