BACKGROUND: Previous studies have explored protective factors against gender minority stress among lesbian, gay, bisexual, transgender, queer, and other gender and sexually diverse (LGBTQ+) individuals. However, many of these studies have relied on self-report data, which may limit the validity of their findings due to potential common method bias. AIM: The present study seeks to understand the biological and psychological underpinnings of the stress-buffering mechanism among LGBTQ+ individuals, specifically examining the protective role of testosterone. METHODS: A total of 107 LGBTQ+ individuals completed a survey on gender-related discrimination, loneliness, and well-being. Additionally, they provided a saliva sample, which was used to derive their endogenous testosterone levels. OUTCOMES: The main outcome was well-being, measured using the World Health Organization-Five Well-Being Index. RESULTS: The findings revealed that gender-related discrimination was associated with heightened levels of loneliness, which in turn were associated with reduced well-being. Testosterone levels significantly moderated the association between gender-related discrimination and well-being. Specifically, among individuals with higher levels of testosterone, the negative association between discrimination and well-being was not significant. CLINICAL IMPLICATIONS: These results suggest that higher levels of testosterone may protect against the psychological consequences of gender-related discrimination. STRENGTHS AND LIMITATIONS: This study represents a pioneering effort to gather empirical evidence on the protective role of testosterone among LGBTQ+ individuals, but it is unlikely that a single biomarker (testosterone) can fully capture the complexity of resilience. Understanding the biological and psychological foundations of minority stress necessitates the integration of multiple biological factors. Such an approach would provide a more comprehensive understanding of the stress-buffering mechanisms operating among LGBTQ+ individuals. CONCLUSION: The study suggests that testosterone may play a significant role in reflecting and regulating the response to gender minority stress.