Estrogen and ovarian function decline are relevant characteristics of menopause period. Numerous physiological, metabolic and immunological alterations in the female body occur in the menopause period and some of these changes remain uncertain. The animal model that mimics menopause phase is an important approach to better comprehend the biological process involved in this period of women life. Ovariectomy is a procedure where ovaries are surgically excised and have been a valuable tool for understanding estrogen deficiency through animal experiments. Despite the diversity of ovariectomy protocols, the aim of this chapter is to provide a comprehensive guideline in performing ovariectomy in mice. Furthermore, isoflurane anesthesia system, postoperative care and surgery success evaluation will be described. We highlight that all procedures must be carried out by a qualified and trained professional, respecting ethical and safety principles.