We aimed to assess the feasibility of passive slow freezing (PSF using Mr. Frosty container, Nalgene) as an alternative to controlled slow rate freezing (CSF using (Freezal™, Air liquide)) for human ovarian tissue (OT) cryopreservation. Validation studies needed were determined after assessing the risk associated (EuroGTP-II ART tool) and were conducted in 66 OT samples from 10 transgender men aged 23.4 ± 5.1 y. Folliculogenesis was assessed in vitro (after 2 h and 2 days of culture) and in vivo (2, 4 and 6 weeks xenotransplantation in Balbc/nude mice) by haematoxilin-eosin staining. Fibrosis was assessed by Masson's trichrome staining. Immunohistochemistry was used to study cell proliferation (PCNA and Ki-67) and apoptosis (caspase-3 and TUNEL). Differences in percentages were estimated using a generalized estimated equations method. After 2 days of in vitro culture, higher odds of primordial follicles (PF) (OR 1.626; 95%CI (1.162–2.266); P = 0.004) and lower odds of growing follicles (GF) (OR 0.616; 95%CI (0.441–0.861); P = 0.004) were associated with the established CSF technique. No statistical differences were found in the mean estimated proportion of proliferating (Ki-67+ or PCNA+) or apoptotic (caspase-3+ or Tunel+) follicles. Two and 6 weeks after xenotransplantation, respectively lower odds of GF (OR 0.419; 95%CI (0.217–0.809); P = 0.010) and secondary follicles (OR 0.135; 95%CI (0.071–0.255); P < 0.001) were associated with CSF. Proportion of fibrosis was similar. This validation study shows a higher follicle activation after 2 days in vitro and after 2 weeks following xenotransplantation in mice using PSF. PSF may be an easy, cost-effective low-risk alternative to CSF for cryopreservation of human OT.