Hydrogels have become one of the most popular platforms for three-dimensional (3D) cultivation of mammalian cells. The enormous versatility of hydrogel materials makes it possible to design scaffolds with predefined mechanical properties, as well as with desired biofunctionality. 3D hydrogel constructs have been used for a variety of applications, including tissue engineering of microorgan systems, drug delivery, cytotoxicity testing, and drug screening. Moreover, 3D culture is applied for investigating cellular physiology, stem cell differentiation, and tumor models and for studying interaction mechanisms between the extracellular matrix and cells. In this paper, we review current examples of performance-based hydrogel design for 3D cell culture applications. A major emphasis is placed on a description of how standard analytical protocols and imaging techniques are being adapted to analysis of 3D cell culture in hydrogel systems.