Lumican is a small leucine-rich proteoglycan in the human amniotic membrane (AM) that promotes corneal epithelialization and the organization of collagen fibers, maintaining corneal transparency. In the present work, a method for protein extraction from AM to obtain lumican is proposed. Additionally, the stability of lumican in the AM extract (AME) stored at different temperatures and time periods is evaluated. 100 mg of AM were thawed and mechanical de-epithelialized. The de-epithelialized AM was frozen and crushed until a fine powder was obtained, which was solubilized with 2.5 mL of saline buffer with protease inhibitors and centrifuged for protein extraction. The supernatant was collected and stored at -20 °C, 4 °C, and room temperature (RT) for 6, 12, 20, and 32 days. Afterward, lumican was quantified in each AME. This technique allows an accessible and acquirable protocol for lumican extraction from AM. Lumican concentration was affected by storage time and temperature conditions. Lumican in the AME of 12 days stored at -20 °C and 4 °C was significantly higher than other AME. This lumican extraction could be useful for developing treatments and pharmaceutical solutions. Further studies are needed to determine the uses of AME lumican in re-epithelialization and wound healing process.