The polar transport of the natural auxins indole-3-butyric acid (IBA) and indole-3-acetic acid (IAA) has been described in Arabidopsis (Arabidopsis thaliana) hypocotyls using radioactive tracers. Because radioactive assays alone cannot distinguish IBA from its metabolites, the detected transport from applied [ 3H]IBA may have resulted from the transport of IBA metabolites, including IAA. To test this hypothesis, we used a mass spectrometry-based method to quantify the transport of IBA in Arabidopsis hypocotyls by following the movement of [ 13C 1]IBA and the [ 13C 1]IAA derived from [ 13C 1]IBA. We also assayed [ 13C 6]IAA transport in a parallel control experiment. We found that the amount of transported [ 13C 1]IBA was dramatically lower than [ 13C 6]IAA, and the IBA transport was not reduced by the auxin transport inhibitor N-1-naphthylphthalamic acid. Significant amounts of the applied [ 13C 1]IBAwere converted to [ 13C 1]IAA during transport, but [ 13C 1]IBA transport was independent of IBA-to-IAA conversion. We also found that most of the [ 13C 1]IBA was converted to ester-linked [ 13C 1]IBA at the apical end of hypocotyls, and ester-linked [ 13C 1]IBA was also found in the basal end at a level higher than free [ 13C 1]IBA. In contrast, most of the [ 13C 6]IAA was converted to amide-linked [ 13C 6]IAA at the apical end of hypocotyls, but very little conjugated [ 13C 6]IAA was found in the basal end. Our results demonstrate that the polar transport of IBA is much lower than IAA in Arabidopsis hypocotyls, and the transport mechanism is distinct from IAA transport. These experiments also establish a method for quantifying the movement of small molecules in plants using stable isotope labeling.