Throughout human history, natural products have been used for crop protection or as medicine and up to today, they play an important role as lead structures in modern drug discovery and development of agrochemicals. Halogenation is a frequently found structural modification that can alter the biological activity of a compound, improve its stability, bioavailability, and selectivity. Benzoxazinoids are a group of indole-derived plant defense compounds with antifeedant, antimicrobial, and allelopathic activities. Compared to plant-derived benzoxazinoids, synthetic halogenated analogs showed improved antifungal and selective herbicidal activity. Chemical total synthesis and derivatization of natural products can be challenging due to poor regioselectivity, complex structural features, or instability of the compound. Production of structural analogs through biosynthetic approaches can help tackle these problems. In a precursor-directed biosynthesis using a heterologous host, analogs of an early pathway intermediate that are commercially available can be incorporated by the enzymes of the pathway. The biosynthesis of benzoxazinoids in plants evolved independently several times and the biosynthetic enzymes involved have been discovered in for several of these plant species. The variety of different enzymes catalyzing the same reaction increases chances for good incorporation of substrate analogs. Starting from halogenated indole, the substrate specificity of the pathway enzymes was characterized individually and the precursor-directed biosynthesis of multiple chlorinated and fluorinated benzoxazinoids in the plant host N. benthamiana was successfully achieved. Furthermore, the combined transient expression of the pathway enzymes together with bacterial halogenases allowed for the complete de novo biosynthesis of halogenated benzoxazinoids in planta. The co-expression of different tryptophan halogenases with a tryptophanase provided chlorinated indole that subsequently was converted into benzoxazinoids. Directing the subcellular localization of the tryptophan halogenases to the chloroplast and thus adapting it to the plant host, led to a significant increase in yields of halogenated benzoxazinoids.