〔Abstract〕 Objective To define hyaluronidase treatment parameters that efficiently remove the extracellular matrix from zygotes without impairing embryo development，thereby providing a basis for standardized assisted reproductive procedures. Methods Mouse zygotes were treated with hyaluronidase at 40，80，or 120 IU/mL for 60，90，or 120 s. Following treatment，embryos were cultured in Krebs-ringer bicarbonate-based solution for organismal media（KSOM）medium，and blastocyst formation was monitored using a Time-lapse imaging system. Results A concentration of 40 IU/mL was insufficient for effective matrix removal，whereas 120 IU/mL markedly reduced blastocyst formation. In contrast，80 IU/mL achieved efficient matrix clearance with minimal impact on development. Blastocyst rates were comparable between the 60 s and 90 s groups，with a slight advantage for the 60 s under the experimental conditions. Conclusion Hyaluronidase treatment at 80 IU/mL for 60 s represents optimal processing conditions for mouse zygotes. These findings provide experimental basis and standardized reference for embryo handling in assisted reproductive technologies.