In the past 20 years, human amniotic membrane (AM) has become widely used as an ophthalmic surgical patch as well as a substrate for stem cell tissue equivalents for ocular surface reconstruction. AM reduces ocular surface scarring and inflammation, and enhances epithelialization. In addition, it shows limited immunogenicity and some anti-microbial properties. Before being applied clinically, the donor of AM is required to undergo a thorough health screening and the membrane has to undergo an accepted processing routine, which includes preservation, sterilization and de-epithelialization. There have been various articles describing methods in preserving, sterilizing and de-epithelializing AM. Each preparation technique has been reported to have differential effects on the physical and biological properties of the AM. Therefore, it is difficult to establish a standardized procedure. In this review, we discuss the present techniques and several novel, new approaches in the preparation of AM for use in ocular surface reconstruction, and their impact on AM structure and biological activity.