Tryptophan and indole derivatives are common precursors in many natural biosynthetic pathways. In recent years it has been shown that the substrate specificity of tryptophan halogenases is much more relaxed than previously thought. Using the tryptophan 7-halogenase PrnA, the tryptophan 6-halogenase ThdH, and the tryptophan 5-halogenase PyrH we achieved the regioselective mono-halogenation of indole-3-acetic acid (IAA), which has not been reported as a substrate of tryptophan halogenases to date. The tryptophan 5-halogenase gene was introduced into Arabidopsis thaliana leading to the formation of 5-chlorotryptophan, 5-chloroindole-3-acetonitrile and 5-chloro-3-indole acetic acid by A. thaliana. PyrH activity could also be demonstrated for the plant-produced halogenase in vitro. These results show the potential of flavin-dependent halogenases to generate novel halogenated auxins or other secondary metabolites in vitro and in vivo by plants.