Biofluorescence, the ability to absorb light and reemit it at a longer wavelength, is present in many taxa but has been examined only recently in amphibians. Over half of the studies examining biofluorescence in the last century suggest this fluorescent signal may affect predation; however, to date, only one experimental study has tested this hypothesis. To address this question, we experimentally tested the effect of biofluorescence on predation through the study of the Cope’s Gray Treefrog, Hyla chrysoscelis. First, we quantified the spectral characteristics of a novel biofluorescence pattern in H. chrysoscelis. In this species, the fluorescent signal is concentrated in an area for both sexes that contains a flash pattern proposed to warn predators of the frog’s toxic secretions. We hypothesized that the biofluorescent trait may increase the conspicuousness of this flash pattern and enable the frogs to deter predators more effectively. Second, we tested this prediction by conducting a clay model field experiment to assess differences in predation attempts on fluorescent versus non-fluorescent H. chrysoscelis models by various predator types. We found no effect of biofluorescence on the overall presence and type of predation, suggesting that biofluorescence does not contribute strongly to the deterring effect of the antipredator flash signal of H. chrysoscelis. This study represents one of the first attempts to experimentally test the effect of biofluorescence on predation in any organism and the first to do so in amphibians. Further work is needed to explore the role of this trait in predation in other systems and to investigate alternative functions for the biofluorescent signal in H. chrysoscelis.