Three-dimensional cell culture techniques and platforms have gained considerable attention in recent years. Owing to the ability to replicate physiologically relevant conditions, these systems appear to be poised to address several challenges posed in tissue engineering, particularly in translational research and application. Despite the widespread acceptance of 3D cultures, techniques to quantitatively assess yields, cell responses, proliferation, and the role of the type of substrate used, addition or absence of adhesion promoters such as collagen, or the method of establishing a 3D cell culture remain a matter of techniques developed in individual laboratories or cell lines. In this special issue, protocols, and methods to establish 3D cultures and techniques to evaluate them will be presented.