ABSTRACT Background Masculinizing hormone therapy with testosterone is used to align an individual’s physical characteristics with their gender identity in trans and gender diverse individuals. Standard testosterone doses and formulations recommended for hypogonadal cisgender men are typically administered. 100 mg AndroForte 5% testosterone cream is the recommended starting dose in hypogonadal cisgender men but there are no data evaluating the use of AndroForte 5% testosterone cream in gender-affirming hormone therapy regimens. Aim To assess the prescription patterns and serum total testosterone concentrations achieved with AndroForte 5% testosterone cream in trans and gender diverse individuals. Methods A retrospective analysis was undertaken of trans and gender diverse individuals at a primary and secondary care clinic in Melbourne, Australia. Seventy-two individuals treated with AndroForte 5% testosterone cream to the torso were included. Outcomes Testosterone dose and serum total testosterone concentration. Results Median age was 26 years (IQR 22–30) and median duration of testosterone therapy was 14 months (7–24). Fifty (69%) individuals had a non-binary gender identity. Initial median testosterone dose was 50 mg (50–100) daily. Thirty-eight (53%) commenced doses <100 mg daily, the recommended starting dose for hypogonadal cisgender men. Median total testosterone concentration achieved from 186 individual laboratory results was 11.9 nmol/L (8.1–16.4). Polycythemia was documented in 5 (7%) individuals. Clinical Implications AndroForte 5% testosterone cream can be used in individuals with a binary and/or non-binary gender identity seeking masculinization. It can be commenced at a lower dose than that administered to hypogonadal cisgender men for individuals seeking slow masculinization goals. Strengths & Limitations Limitations include the retrospective study design, lack of clinical end points and lack of standardization of timing of laboratory tests in relation to the last dose. This is the first study to evaluate AndroForte 5% testosterone cream in trans and gender diverse individuals and provides insights into prescription patterns in individuals with a non-binary gender identity. Conclusion AndroForte 5% testosterone cream represents an alternative formulation of testosterone administration for trans and gender diverse individuals seeking masculinization.