Folliculogenesis is a complex and precisely regulated physiological process. Performing a complete in vitro folliculogenesis remains challenging and has not been yet achieved in the sheep. Successfully accomplishing folliculogenesis in a large animal such as the sheep is an essential step in validating in vitro folliculogenesis technique and its further application to fertility preservation in human. The main knowledge gap hindering this achievement is a better comprehension of mechanical and biochemical signals involved in the initiation of follicular growth from the reserve pool of primordial follicles. Here, we describe the dissection and stretching methods as well as the in vitro culture of sheep ovarian cortex tissue in view to initiate folliculogenesis.