Thirty years ago, an explanation for the facultative increase of males’ androgen levels in response to social challenges was proposed as the “Challenge Hypothesis” (Wingfield et al., 1990). Since then, numerous studies have tested this hypothesis across different animal taxa with diverse life history. We tested the Challenge Hypothesis in the highly territorial poison frog, Allobates femoralis. We compared males’ androgen concentrations between a non-stimulated condition (baseline) and following a simulated territorial intrusion (post-STI) conducted by playing back the territorial call through a loudspeaker. We took advantage of water-borne hormones sampling, a non-invasive technique, to characterize androgen levels, and showed that it closely reflects circulating plasma testosterone levels. Our results demonstrate that water-borne androgen increases after a STI in A. femoralis males only when males approached the playback loudspeaker. Therefore, our results provide novel support to the Challenge Hypothesis in a territorial frog.