Hair steroid analysis is increasingly recognized for its ability to capture cumulative hormone secretion, thought to reflect an individual's response to long-term environmental conditions. Despite its growing use, the influence of single, isolated events on hair steroid concentrations remains underexplored. Our study therefore examined the effects of a single-dose transdermal testosterone administration (150 mg) and acute laboratory stress induction on hair testosterone and cortisol levels in a randomized, between-subject, placebo-controlled, and double-blind design. Liquid chromatography-tandem mass spectrometry analysis revealed a significant increase in hair testosterone levels seven weeks post-testosterone administration, underscoring the efficacy of hair analysis in detecting single-use hormone intake. Moreover, we observed a significant elevation in hair cortisol levels seven weeks post-exposure to the laboratory somatic stressor (Cold pressor test), highlighting the efficacy of hair analysis in capturing experimentally induced hormonal responses. The exploration of contextual factors revealed that individuals in committed relationships exhibited lower levels of both hair cortisol and testosterone compared to those who were single or in uncommitted relationships. Our study provides new insights into the sensitivity of hair analysis for detecting hormonal changes following single-dose hormone administration and experimentally induced short-term stress events. The exploratory findings emphasize the importance of individual contextual factors in influencing hair hormone concentrations and lay the groundwork for further investigation into the dynamics of cumulative hair hormone measurements.