〔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.