This paper provides a summary of the most important chemical and physical parameters of tears that can help the formulator in the development of new ocular formulations and in the conception of innovative ophthalmic delivery approaches. For each physiological parameter, the relevance in ocular drug delivery is discussed in detail and the analytical tools that are used for the determination of these parameters are described and summarized. The aim of this review is also to give a description of the main analytical techniques available in ophthalmology that can be used for pharmacokinetic studies of active compounds. The importance of tear sampling techniques used in the determination of the parameters is also discussed.