Introduction: Gender-affirming hormone treatment is known to affect adrenal androgen levels in adult individuals with gender dysphoria (GD). This may be clinically relevant because the adrenal gland plays a critical role in many different metabolic processes. Aim: This study aims to assess the effects of gonadotropin-releasing hormone analogs (GnRHa) treatment and gender-affirming hormone treatment on adrenal androgen levels in adolescents with GD. Methods: In this prospective study, dehydroepiandrosterone-sulfate (DHEAS) and androstenedione values were measured every 6 months during 2 years of GnRHa treatment only, and 2 years of GnRHa combined with gender-affirming hormone treatment (estradiol or testosterone) in 73 transgirls and 54 transboys. To determine trends in adrenal androgen levels a linear mixed model was used to approximate androgen levels. Main Outcome Measures: DHEAS and androstenedione levels were the main outcome measures. Results: DHEAS levels rose in transboys during GnRHa treatment, which may represent the normal increase during adolescence. In transgirls no change in DHEAS levels during GnRHa treatment was found. Gender-affirming hormone treatment did not affect DHEAS levels in either sex. In transboys androstenedione levels decreased during the first year of GnRHa treatment, which may reflect reduced ovarian androstenedione synthesis, and rose during the first year of gender-affirming hormone treatment, possibly due to conversion of administered testosterone. In transgirls androstenedione levels did not change during either GnRHa or gender-affirming hormone treatment. Clinical Implications: No deleterious effects of treatment on adrenal androgen levels were found during approximately 4 years of follow-up. Strengths & Limitations: This is one of the largest cohort of adolescents with GD, treated using a uniform protocol, with standardized follow-up. The lack of a control group is a limitation. Conclusion: The changes in androstenedione levels during GnRHa and gender-affirming hormone treatment in transboys may not be of adrenal origin. The absence of changes in androstenedione levels in transgirls or DHEAS levels in either sex during gender-affirming hormone treatment suggests that gender-affirming hormone treatment does not significantly affect adrenal androgen production. Schagen SEE, Lustenhouwer P, Cohen-Kettenis PT, et al. Changes in Adrenal Androgens During Puberty Suppression and Gender-Affirming Hormone Treatment in Adolescents With Gender Dysphoria. J Sex Med 2018;15:1357–1363.