Objective: Hyaluronidase enzyme is an extremely important factor for the process of oocyte denudation, but little is known about its negative effects.Methods: This prospective randomized study analyzed the results of using different concentrations of hyaluronidase (Diluted: 8IU/mL and Normal: 80IU/mL) used for denudation of sibling-oocytes for 22 women undergoing treatment for assisted reproduction by ICSI. A total of 192 oocytes were injected, being 104 for group I (diluted) and 88 for group II (normal). We analyzed fertilization rate, cleavage, embryo quality at 48 and 72 hours and number of transferred embryos in each group. Results: The diluted enzyme group showed better results in fertilization rates (92.3% vs. 80.6%), mean cleavage (4.18 ± 2.57 vs. 3.09 ± 1.90), in 48-hour embryos A and A + B (60.9% vs. 44.1% and 90.2% vs. 82.3%) and at 72 hours (45.6% vs. 36.8% and 77.1% vs 66.2%), and number of embryos selected for transfer (61.8% vs. 38.1%). The overall pregnancy rate was 59.1%. Conclusion: This study demonstrates that the use of 8 IU/mL of hyaluronidase, according to the following protocol, is beneficial and can be successfully used for oocyte denudation, and it is also economically advantageous to the laboratory.