Scaffolds play a critical role in tissue engineering (TE), in particular scaffold-based TE, and they are designed to be biocompatible, with a suitable degree of porosity and surface chemistry to allow attachment, migration, proliferation, differentiation, and infiltration of the cells. Other important characteristics of scaffolds include having adequate mechanical properties, controlled biodegradability, and the ability to mimic in part the structure and biological function of the extracellular matrix. Keeping these key points in mind, this chapter focuses on the protocols for the preparation and characterization of conventional and novel scaffold biomaterials for the application of TE utilizing several synthetic and natural polymers. © 2012 Scrivener Publishing LLC. All rights reserved.