The objective of the current study was to use a biomarker-based approach to investigate the influence of atrazine exposure on American toad (Anaxyrus americanus) and grey tree frog (Hyla versicolor) tadpoles. Atrazine is one of the most frequently detected herbicides in environmental matrices throughout the United States. In surface waters, it has been found at concentrations from 0.04–2859 μg/L and thus presents a likely exposure scenario for non-target species such as amphibians. Studies have examined the effect of atrazine on the metamorphic parameters of amphibians, however, the data are often contradictory. Gosner stage 22–24 tadpoles were exposed to 0 (control), 10, 50, 250 or 1250 μg/L of atrazine for 48 h. Endogenous polar metabolites were extracted and analyzed using gas chromatography coupled with mass spectrometry. Statistical analyses of the acquired spectra with machine learning classification models demonstrated identifiable changes in the metabolomic profiles between exposed and control tadpoles. Support vector machine models with recursive feature elimination created a more efficient, non-parametric data analysis and increased interpretability of metabolomic profiles. Biochemical fluxes observed in the exposed groups of both A. americanus and H. versicolor displayed perturbations in a number of classes of biological macromolecules including fatty acids, amino acids, purine nucleosides, pyrimidines, and mono- and di-saccharides. Metabolomic pathway analyses are consistent with findings of other studies demonstrating disruption of amino acid and energy metabolism from atrazine exposure to non-target species.