The eye is a complex organ composed of multiple tissues in anterior and posterior eye segments. Malfunctions of any of these tissues can lead to ocular diseases and loss of vision. A detailed understanding of the ocular anatomy and physiology in animal models and humans contributes to the development of ocular drugs by enabling studies on drug delivery and clearance routes, pharmacokinetics, and toxicity. This protocol provides step-by-step instructions for the extraction and homogenization of ocular tissues for enzymatic and proteomics analyses. Key features • Suitable protocol for the extraction and isolation of ocular tissue from humans and laboratory animals (rabbit, pig, rat, mouse) while minimizing cross-contamination. • Hard or soft tissue homogenates can be prepared efficiently using a Bead Ruptor homogenizer. • Allows to determine the protein contents in prepared homogenates.