AIM: To experiment the toxicity of protoplast induced enzyme on human corneal stromal cells in vitro. METHODS: Protoplast induced enzyme included 1g/dL snailase, 1g/dL cellulase and 0.1g/dL lysing enzyme. The enzyme and human corneal stromal cells were cultured for 15 minutes, 4 hours, 8 hours. The effect of enzyme on the human corneal stromal cells was experimented with MTT assay and trypan blue staining. RESULTS:The cell morphology did not change after 15 minutes and 4 hours. The intercellular space of cultured cells was a little smaller after 8 hours, and only to see a few dead cells. MTT value was not noticeable decline after 8 hours. Trypan blue staining showed less impact on cell survival by the enzyme within 8 hours. CONCLUSION: Protoplast induced enzyme showed scarce toxicity on cultured human corneal stromal cell within a short time. The concentration of the enzyme used in future experimental animal models and human cytology experiment is safe.