Context <p>Ovarian tissue autotransplantation is currently the only proven technique for reusing ovarian tissue after fertility preservation by ovarian tissue cryopreservation. However, one of its limitations relates to the quality of the grafts, both in terms of the number of surviving follicles and the quality of the stromal environment, which is essential for follicular development and graft revascularization. The aim of this study was to validate a technique for characterizing and functionally qualifying ovarian tissue in order to identify cell sub-populations of interest.</p> Materials <p>Ovarian cortex strips were collected during ovarian drilling in women suffering from polycystic ovary syndrome. After fresh or frozen ovarian tissue dissociation, the resulting ovarian cells were analyzed by multicolor flow cytometry (MFC) to determine cell yield and viability after dissociation, and to identify for specific with specific antibodies.</p> Results <p>Yield was significantly higher after dissociation of fresh ovarian tissue (1,59 × 10⁶ viable nucleated cells per 100&#xa0;mg of ovarian cortex) compared with frozen/thawed ovarian tissue ((1,08 × 10⁶ viable nucleated cells per 100&#xa0;mg of ovarian cortex) (<i>p</i> = 0,0195). Conversely, viability was significantly higher after dissociation of frozen/thawed ovarian tissue (84,7%) compared with fresh ovarian tissue (84,4%) (<i>p</i> = 0,0367). Using a panel of antibodies enabled the identification of different sub-populations that could correspond to endothelial cells or progenitors, cells with a mesenchymal profile and pericytes.</p> Conclusion <p>Although further panel development is required, MFC effectively characterizes cell populations within ovarian tissue. Non-follicular cells could be evaluated as a potential prognostic factor for the recovery of ovarian function after autotransplantation but also participate in ovarian reconstruction programs.</p>