Purpose <p>Laser-assisted hatching (LAH) is a typical procedure in ART. It is used when embryos are unable to hatch. To reduce the potential damage to embryos caused by millisecond laser typically used to create <i>zona pellucida</i> openings, we have developed a protocol for femtosecond laser microsurgery. The aim of this study was to investigate the possible effects of the femtosecond laser, as opposed to the millisecond dissector used in LAH.</p> Methods <p>Zygotes obtained from hormonally stimulated wild-type mice were cultured in vitro until blastocyst stage. Two types of lasers were used to perform LAH: femtosecond and millisecond. The femtosecond laser system used in this study was a TETA ytterbium laser. As a millisecond laser, we used the Octax Laser Shot dissector (pulse duration 5 and 10ms).</p> Results <p>To analyze whether the femtosecond laser could be dangerous for embryos, we evaluated the presence of reactive oxygen species (ROS) and heat-shock proteins (HSP). We found no differences in the production of ROS and in the synthesis of HSP at both the protein and mRNA levels between the negative control and the femtosecond LAH groups, whereas ROS levels in the millisecond LAH group of embryos were close to those observed in the positive control group. Millisecond laser-treated embryos collapsed, while embryos from the femtosecond LAH group did not exhibit such morphological changes.</p> Conclusions <p>Application of femtosecond laser for LAH procedure is effective and does not lead to the production of HSP and ROS in embryos.</p>