This dissertation describes the engineering of halogenating enzymes (halogenases) to facilitate the site-selective functionalization of bioactive molecules. While most small-molecule methods of halogenation have selectivities dictated by the electronics of the substrate, there exist enzymes that are able to functionalize (an albeit limited set of) substrates at significantly electronically disfavored positions. Prior to the work described within this dissertation, the extremely limited protein engineering efforts performed on these enzymes had been unable to increase their activities on substrates larger than their native substrates. The work described herein entails the first such engineering of one halogenase in particular, RebH, to allow the functionalization of a range of large, bioactive substrates with selectivities not observed with small-molecule strategies. Chapter I provides a background to halogenases and biocatalysis. Halogenation is a vital process in the production of a significant percentage of pharmaceuticals and agrochemicals, both to facilitate chemical synthesis and to tailor the biological properties of the products. Despite this, there is still a lack of methodology to install halogen substituents at electronically disfavored positions on many substrates of interest. A number of halogenases, some with selectivities for electronically disfavored positions, have been reported and characterized, and in this chapter a summary of the existing halogenase literature will be provided. A unique advantage of using enzymes as catalysts rather than small molecules is the ability to genetically introduce mutations to alter the properties of your catalyst; by doing so iteratively, one can recapitulate natural selection toward the goals of the researchers on laboratory timescales in a process known as directed evolution. While this process had not been applied to halogenases prior to the work described in this dissertation, it is a well-developed technique with years of use and success. A brief introduction to directed evolution will also be provided in this chapter as a prelude to the description of its application to halogenases. Chapter II describes the enhancement of the expression of soluble RebH in E. coli and the exploration of the scope of wild-type RebH. A number of halogenases were explored in E. coli and RebH was selected for efforts to improve its solubility. Once this was accomplished, it allowed wild-type RebH to be tested on preparative scales on a range of unnatural substrates. Activity on several unnatural substrates was observed with high selectivity for electronically disfavored positions. The work described in this chapter was necessary to allow the directed evolution described in the following chapter. Chapter III describes the directed evolution of RebH. The improvement of the thermostability of RebH by directed evolution will be briefly summarized, which provided the first step of the expansion of the substrate scope of RebH toward large, bioactive molecules. The directed evolution of RebH to expand its substrate scope, as well as preparative-scale bioconversions of a range of important bioactive substrates and kinetic analysis of the evolution lineage, will then be described. The engineered RebH variants developed in this process have proven to be broadly useful for a diverse range of substrates, and the screening of a library of compounds will be described to illustrate this point. Lastly, halogenation provides for a useful synthetic handle for further functionalization, and the development of a facile procedure for subsequent cross-coupling of crude products from bioconversions will be briefly discussed. Chapter IV describes the use of RebH variants to perform enantioselective halogenations via desymmetrization on a range of symmetric substrates. Symmetric, prochiral dianiline substrates were synthesized, halogenated using the engineered RebH variants, the development of which was described in Chapter III, and the monochlorinated products found to be enantioenriched. It was then demonstrated that protein engineering can be used to alter the yield and enantioselectivity of these bioconversions. The work described in this last chapter marks the first reported application of this class of enzymes in enantioselective catalysis.