Alongside biosynthesis and transport, inactivation regulates indole-3-acetic acid (IAA) concentration, a compound with numerous functions in plant development. The main inactive IAA metabolites are oxidised forms and ester- or amide-linked conjugates. DIOXYGENASE FOR AUXIN OXIDATION1 (DAO1) and DAO2, 2-oxoglutarate and iron-dependent dioxygenases, contribute to IAA oxidative inactivation with group II GRETCHEN HAGEN3 (GH3) IAA-amido synthetases, while UDP-glycosyltransferases (UGTs) conjugate IAA to sugars. To study IAA inactivation routes, we generated combinatorial Arabidopsis mutants between all group II GH3s (gh3oct) and DAO1 or DAO2, and between DAOs and the main UGTs. In vivo [13C6]IAA feeding experiments traced exogenously applied IAA’s metabolic fate, supporting the main IAA inactivation pathway where DAOs act downstream of GH3s. Results from these experiments also indicated UGT-mediated IAA glycosylation is more important than previously assumed for modulating IAA levels and plant development. Our metabolic and transcriptomic data revealed that gh3oct may still retain some GH3 activity, explaining previously reported phenotypic inconsistencies. Our data additionally suggest that unidentified metabolic activities might play a role in IAA overproducing plants, and that premature downregulation of flowering time regulators like FLOWERING LOCUS C (FLC) likely underlies early flowering of gh3oct and gh3oct dao1-6 plants.