A corneal stromal lenticule (CSL) is a part of the corneal stroma that forms as a waste product during the refractive surgery ReLEx SMILE (Small Incision Lenticule Extraction) and is no longer used. With the increasing number of ReLEx SMILE procedures and the number of potentially available CSLs, two main aspects of their usage are currently being investigated. The first aspect includes the biological properties of CSLs and their proper preservation with respect to long-term storage. The second aspect is related to the potential clinical use of CSLs. As a high-quality biomaterial, CSLs have substantial potential to be used for alternative solutions in the treatment of specific eye diseases. In a number of studies it has been shown that RSL transplantation could be a safe and effective method that does not cause any serious complications, for example in terms of immune reaction. The aim of this article is to present an overview of the possibilities for using CSLs for transplantation purposes, and at the same time to discuss our methodology for processing and preserving CSLs with the protocol used at the Eye Tissue Bank of the Královské Vinohrady University Hospital.