In this chapter, the optimization of the design of vitreous substitutes will be discussed. First, a review will explain the structure and function of the vitreous humor and the need for a better vitreous substitute. The approach taken in this work will be to develop a biomimetic vitreous substitute. As a result, the next section will discuss the biomechanics of the natural vitreous humor and the development of an animal model for the ideal vitreous substitute. Mixture design is used to screen candidates rapidly for use as in situ-forming polymeric hydrogel vitreous substitutes. Statistical experimental design is used to determine the effects of the crosslinker content, hydrophobic substitution, and polymer concentration in the hydrogel on the optomechanical properties. In addition, the equilibrium swelling properties are characterized and the osmotic pressure exerted by the hydrogel is calculated to determine whether or not these hydrogels could tamponade the retina via exertion of a slight osmotic pressure as vitreous substitutes. 2010 Woodhead Publishing Limited All rights reserved.