Abstract Background Twenty years ago, the Dutch Protocol—consisting of a gonadotropin-releasing hormone agonist (GnRHa) to halt puberty and subsequent gender-affirming hormones (GAHs)—was implemented to treat adolescents with gender dysphoria. Aim To study trends in trajectories in children and adolescents who were referred for evaluation of gender dysphoria and/or treated following the Dutch Protocol. Methods The current study is based on a retrospective cohort of 1766 children and adolescents in the Amsterdam Cohort of Gender Dysphoria. Outcomes Outcomes included trends in number of intakes, ratio of assigned sex at birth, age at intake, age at start of GnRHa and GAH, puberty stage at start of GnRHa, proportions of adolescents starting and stopping GnRHa, reasons for refraining from GnRHa, and proportions of people undergoing gender-affirming surgery. Results A steep increase in referrals was observed over the years. A change in the AMAB:AFAB ratio (assigned male at birth to assigned female at birth) was seen over time, tipping the balance toward AFAB. Age at intake and at start of GnRHa has increased over time. Of possibly eligible adolescents who had their first visit before age 10 years, nearly half started GnRHa vs around two-thirds who had their first visit at or after age 10 years. The proportion starting GnRHa rose only for those first visiting before age 10. Puberty stage at start of GnRHa fluctuated over time. Absence of gender dysphoria diagnosis was the main reason for not starting GnRHa. Very few stopped GnRHa (1.4%), mostly because of remission of gender dysphoria. Age at start of GAH has increased mainly in the most recent years. When a change in law was made in July 2014 no longer requiring gonadectomy to change legal sex, percentages of people undergoing gonadectomy decreased in AMAB and AFAB. Clinical Implications A substantial number of adolescents did not start medical treatment. In the ones who did, risk for retransitioning was very low, providing ongoing support for medical interventions ...