To study the impact of unilateral ovariectomy for ovarian tissue cryopreservation (OTC) on the function of the remaining ovary in girls with Turner syndrome. A prospective cohort study as follow up of OTC in a research setting (The TurnerFertility trial, NCT03381300). University hospital in Nijmegen, the Netherlands. A total of 28 girls with Turner syndrome with follicles in their cryopreserved ovarian cortex tissue, aged 5-19 years. Of the 28 girls, 21 girls had a 45,X/46,XX mosaic karyotype, five had structural aberrations of the X-chromosome, one girl had a 45,X monosomy, and one girl had a 45,X/47,XXX karyotype. Girls were monitored annually after OTC for pubertal development and levels of anti-Müllerian hormone (AMH), Follicle Stimulating Hormone, Luteinizing Hormone, Estradiol, and Inhibin B. Thelarche, menarche and onset of premature ovarian insufficiency. The girls were monitored for a median duration of 3.4 years (maximum 6.6 years). The pubertal development of five prepubertal girls is still unknown, all were under the age of 10 and had low gonadotropins and estradiol levels at the end of the follow-up. Seven of the eight girls around pubertal age (10-12 years) experienced spontaneous thelarche, while one received medication to induce puberty. Eleven of the fourteen girls between the age of 14-17 years experienced spontaneous menarche, three other girls with thelarche still had ongoing puberty at the end of follow-up with normal gonadotropins and AMH levels above the detection limit. Around six to twelve months after OTC, a decline in AMH concentration was observed in 57% of girls (16/28 girls), followed by an increase in AMH concentration in the following years. Six of the total 28 girls started hormone replacement therapy due to symptoms of premature ovarian insufficiency, and all had AMH levels below 0.50 μg/L before OTC. Pubertal development progressed after unilateral ovariectomy for OTC in most girls with Turner syndrome. Hormone replacement therapy was required within a few years for girls with unfavorable parameters before OTC, such as AMH below 0.50 μg/L. Decisions regarding OTC should be personalized, considering the girl's preferences and specific characteristics.