Microcomputed tomography (µ-CT) is a nondestructive, high-resolution, three-dimensional method of analyzing objects. The aim of this study was to evaluate the feasibility of using µ-CT as a noninvasive method of evaluation for tissue-engineering applications. The polyurethane aortic heart valve scaffold was produced using a spraying technique. Cryopreserved/thawed homograft and biological heart valve were decellularized using a detergent mixture. Human endothelial cells and fibroblasts were derived from saphenous vein segments and were verified by immunocytochemistry. Heart valves were initially seeded with fibroblasts followed by colonization with endothelial cells. Scaffolds were scanned by a µ-CT scanner before and after decellularization as well as after cell seeding. Successful colonization was additionally determined by scanning electron microscopy (SEM) and immunohistochemistry (IHC). Microcomputed tomography accurately visualized the complex geometry of heart valves. Moreover, an increase in the total volume and wall thickness as well as a decrease in total surface was demonstrated after seeding. A confluent cell distribution on the heart valves after seeding was confirmed by SEM and IHC. We conclude that µ-CT is a new promising noninvasive method for qualitative and quantitative analysis of tissue-engineering processes.