Posterior segment drug delivery challenges inherent in the treatment of many sight-threatening diseases have become increasingly apparent. Therapeutic interventions for ocular diseases such as neovascular retinopathies, inflammatory and/or infectious diseases may involve drug delivery to vitreoretinal targets. An important part of successful therapeutic strategies for such diseases involves verification that efficacious concentrations of the pharmacological agent are achieved within relevant intraocular regions. Microdialysis has been effectively employed for characterizing intraocular disposition in both anterior and posterior segments, providing important documentation of successful drug delivery to desired targets. Recent papers that showcase the maturation in the model development of microdialysis approaches for estimating posterior segment pharmacokinetics and further validation of the methodology are described in this review. Special problems examined include anterior and posterior ocular clearance mechanisms, intraocular metabolism and active transport of drugs.