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Enzyme engineering by directed evolution presents a powerful strategy for tailoring the function and physicochemical properties of biocatalysts to therapeutic and industrial applications. Our laboratory's research focuses on developing novel molecular tools for protein engineering, as well as on utilizing these methods to customize enzymes and to study fundamental aspects of their structure and function. Specifically, we are interested in nucleoside and nucleotide kinases which are responsible for the intracellular phosphorylation of nucleoside analog (NA) prodrugs to their biologically active triphosphates. The high substrate specificity of the cellular kinases often interferes with prodrug activation and consequently lowers the potency of NAs as antiviral and cancer therapeutics. A working solution to the problem is the co-adminstration of a promiscuous kinase from viruses, bacteria, and other mammals. However, further therapeutic enhancements of NAs depend on the selective and efficient prodrug phosphorylation. In the absence of true NA kinases in nature, we are pursuing laboratory evolution strategies to generate efficient phosphoryl-transfer catalysts. This review summarizes some of our recent work in the field and outlines future challenges.