Few studies investigate the impact of psychosocial stress on acute testosterone activity in adolescents, despite theoretical understandings that testosterone’s relationship with aggressive behavior is dependent upon stress or challenge contexts. Effective administration of laboratory stress, biospecimen collection and storage protocol, and testosterone assaying processes are all important methodological considerations that may explain why the link between testosterone and acute stress response remains unclear in this population. Additionally, the biopsychosocial factors that may predict acute testosterone reactivity in this population also remains unknown. As such, the current study addresses these gaps by examining testosterone activity (via salivary samples) across the administration of a Trier Social Stress Test in a sample of 88 adolescents (46.4% female, Mage = 13.91 years, SDage = 1.57). A series of nine biopsychosocial factors (age, gender, pubertal status, affective reactivity, prosocial behaviors, conduct problems, emotional and behavioral regulation, and exposure to early life adversity) were examined as potential predictors of (1) overall testosterone levels and (2) acute changes of testosterone across the administration of laboratory stress. Results indicated a robust response to psychosocial stress, supporting the notion that testosterone mounts an acute stress response in adolescents. However, only age and sex uniquely predicted overall testosterone and acute reactivity to stress in this sample. These findings suggest a methodological framework for capturing testosterone reactivity to acute stress, and provide evidence for future research to clarify the role of testosterone reactivity in adolescent behavior.