Hydrogels can be used in many biomedical applications and can be imprinted with hydrophilic target molecules through hydrogen bonding. The imprinting process leaves a void space within the hydrogel matrix that includes both the shape and the functionality of the target molecules, thus enabling a thermodynamically favorable interaction between the target molecules and the hydrogel matrix. Our laboratory has developed a standard method for imprinting hydrophilic drug molecules into the hydrogel structure. While applications may include both drug delivery and signal recognition in chemical sensors, we report here on applications in drug delivery and the therapeutic treatment of ocular diseases by utilizing timolol maleate as the target molecule. Here, we describe the protocols we have developed for the preparation of molecular imprinted hydrogels for use in drug delivery applications.