Impaired hatching of the embryo from the zona pellucida (ZP), the specialized protective shell, immediately before implantation is one of the factors of infertility. Hatching impairment is often due to the ZP hardening or thickening. In such cases, the laser assisted hatching procedure is used to overcome infertility. During this procedure a hole is drilled in the ZP facilitating the embryo release. The question of the safe use of laser for assisted hatching remains open, since laser beam can heat the environment and cause thermal shock in embryos. The study was aimed to assess safety of the mouse embryo femtosecond laser exposure during the assisted hatching procedure using the embryo viability and HSP gene expression assessment methods. A new type of pulsed laser was used in the study for the ZP dissection — the femtosecond laser. The energy of such pulses was two orders of magnitude lower than the energy of laser dissectors currently used in the clinics. To assess the femtosecond laser exposure to the embryo, the house mouse (Mus musculus) embryos were stained with fluorescence dyes, and expression of the genes encoding heat shock proteins (Hsp90aa1 and Hspa5) was assessed. The embryonic cells remained viable after the laser assisted hatching procedure involving the use of a femtosecond laser, while expression levels of the genes encoding heat shock proteins were slightly increased compared to the negative control group (p = 0.408).