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Purpose:The purpose of this study was to compare the clinical acceptability of testosterone levels, time to treatment, and postinstruction questions/problems between in-person and telehealth injection teaching for adolescents and young adults (AYA) initiating gender-affirming testosterone therapy.Methods:A retrospective cohort study was conducted using electronic medical record data from a large pediatric gender health clinic. Patients who received subcutaneous testosterone prescriptions between March 15, 2018, and March 14, 2022, were included. The study compared patients receiving in-person versus telehealth injection teaching. Data were collected on demographics, testosterone levels at 3 and 6 months, time from prescription to treatment, and post-instruction contacts. Statistical analyses included t-tests, chi-square tests, logistic regression, linear regression, and zero-inflated negative binomial.Results:The study included 278 patients, with 136 (48.9%) receiving in-person teaching and 142 (51.1%) receiving telehealth teaching. There were no significant differences in baseline characteristics between groups. Clinical effectiveness, indicated by testosterone levels at 3 and 6 months, revealed no significant difference between instruction methods (p= 0.768 and p= 0.350). Time to treatment initiation was comparable (in-person: 15.34 days; telehealth: 18.02 days), with no significant difference in adjusted analysis (p= 0.204). Post-instruction contacts were slightly higher in the telehealth group (2.87 vs. 2.42, p= 0.040), but injection-related questions were rare and similar between groups (p= 0.650).Conclusion:Telehealth instruction for testosterone injection is as effective as in-person teaching methods for AYA initiating gender-affirming care. The findings support the continued use of telehealth to enhance access to gender-affirming care, particularly in light of its clinical efficacy and patient acceptability.