The disadvantages of human amniotic membrane (hAM), used for ocular surface reconstruction, necessitate the development of standardized alternatives. Keratin-derived-films (KF) have been indicated as transferable substrate for cell cultivation and tissue engineering. The impact of different sterilization procedures on KF and surgical feasibility were investigated. Human hair KF were prepared and sterilized; optical, biomechanical properties, in vitro cell seeding efficiency and proliferation of human corneal epithelial cells were studied and compared with hAM. Surgical feasibility was tested on enucleated porcine eye. Sterilized KF showed higher light transmission and significantly higher E-modulus than hAM; cell-seeding-efficiency and proliferation rate were not affected. Although KF could be surgically handled, suture placement was more difficult compared to hAM. Plasma treatment seems the best sterilization method for KF; it does not affect cell biology or optical and biomechanical properties. However material modifications are requested before KF may represent a feasible alternative for ocular surface reconstruction.