Gradient hydrogels represent a pivotal and expanding direction of three-dimensional cell culture. Since various types of gradients play an important role in physiological and pathological processes in vivo, recreation of these gradients in vitro allows a better understanding of cellular behavior, intercellular and cell-matrix interactions. Moreover, gradient hydrogels can advance the creation of functionally improved and physiologically relevant tissue engineered constructs. Another application of gradient hydrogels is the optimization of the 3D in vitro microenvironment (e.g., in terms of hydrogel stiffness or concentration of adhesion ligands). Tunable hydrogels provide researchers with a versatile toolbox to manufacture such gradients in vitro. In this chapter different types of in vivo and in vitro gradients in hydrogels will be presented. Equipment and methods for various gradient fabrications will be discussed. Furthermore, methods of gradient characterizations in hydrogels will be reported. As one of the most recent developments, the influence of low oxygen concentration on cells, as well as the creation and characterization of oxygen gradients in hydrogels will be described. In the last part, achievements in the creation of multiple combinatorial gradients will be presented. The aim of this chapter is to give the reader an overview on existing techniques and biological importance of gradient hydrogels in basic science as well as in applied research.