A modified version of the Trier Social Stress Test (TSST) was employed to determine whether exposure to a virtual audience using virtual reality (VR) technology would prompt an increase of neuroendocrine activity comparable to that prompted by a real audience. Following an anticipatory period, participants completed a speech or a speech-plus-math challenge in front of either a virtual audience, a panel of judges they were led to believe was behind a one-way mirror, or an audience comprised of confederates. An additional group that had prepared a speech was simply directed to observe the virtual audience but did not deliver the speech. Finally, a control group completed questionnaires for the duration of the experiment. Cortisol samples were obtained upon arrival to the laboratory, just before the challenge, and 15 and 30 minutes after the task. Participants also completed a measure assessing stressor appraisals of the task before and after the challenge. Anticipation of the task was associated with a modest increase of cortisol levels, and a further rise of cortisol was evident in response to the challenge. The neuroendocrine changes evoked by the virtual audience were comparable to those elicited by the imagined audience (behind the one-way mirror) but less than changes evoked by the panel of confederates. Stressor appraisals were higher post-challenge compared to those reported prior to the task; however, appraisals were similar across each group. These data suggest that VR technology may be amenable to evaluating the impact of psychosocial stressors such as the TSST.