Addressing some of the most challenging problems that we face today, including depletion of natural resources, sustainable energy production and the generation of green polymeric materials by the biocatalytic upcycling of renewable synthons, requires an expansion of the current available biochemical reaction space. Creating biocatalysts harboring novel chemistries – whether inside or outside the cell – is dependent on the discovery of novel enzymes and metabolic pathways, together with the de novo design of enzymes and directed evolution. Herein we review the high potential of using bioinformatics and in silico computer modelling tools to guide protein engineering and to enhance our fundamental understanding of biocatalysis. Following an overview of technical considerations and the current state-of-the art in sequence- and structure-based protein engineering methodologies, we highlight recent successful examples of their implementation in biocatalysis and synthetic biology. Moreover, we discuss how selected computational tools in concert with experimental biocatalysis could decipher how the sequence, structure and dynamics of proteins dictate their function. Using the methodologies discussed in this chapter, an accelerated biocatalytic manufacturing of chemicals, pharmaceuticals, biofuels and monomeric building blocks is envisioned.