The unique anatomy and physiology of the eye present many challenges to the successful development and delivery of ophthalmic drugs. Any therapeutic strategy developed to control the progression of anterior and posterior segment diseases requires continuous monitoring of effective drug concentrations in the relevant ocular tissues and fluids. Ocular microdialysis has gained popularity in recent years due to its ability to continuously monitor drug concentrations and substantially reduce the number of animals needed. The intrusive nature of ocular microdialysis experimentation has restricted these studies to animal models. This review article intends to highlight various aspects of ocular microdialysis and its relevance in examining the disposition of drugs in the anterior and posterior segments.