BACKGROUND: Increasing numbers of transgender and gender-diverse individuals are seeking initiation of gender-affirming hormone therapy. This aligns an individual's physical characteristics with their gender identity and improves psychological outcomes. Physical changes, including changes to muscle mass and body fat redistribution, can alter sex-specific laboratory reference ranges. CONTENT: We review the impact of gender-affirming hormone therapy on laboratory parameters with sex-specific reference ranges, with a focus on hemoglobin/hematocrit, renal function, cardiac biomarkers, and prostate-specific antigen. SUMMARY: Gender-affirming hormone therapy results in changes in laboratory parameters with sex-specific reference ranges. For individuals established on gender-affirming hormone therapy, reference ranges that align with an individual's gender identity should be used for hemoglobin/hematocrit, serum creatinine, and high-sensitivity cardiac troponin and N-terminal brain natriuretic peptide. Clinicians should interpret these biomarkers according to the reference range that aligns with one's affirmed gender.