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In vitro biotransformation (ivBT) refers to the use of an artificial biological reaction system that employs purified enzymes for the one-pot conversion of low-cost materials into biocommodities such as ethanol, organic acids, and amino acids. Unshackled from cell growth and metabolism, ivBT exhibits distinct advantages compared with metabolic engineering, including but not limited to high engineering flexibility, ease of operation, fast reaction rate, high product yields, and good scalability. These characteristics position ivBT as a promising next-generation biomanufacturing platform. Nevertheless, challenges persist in the enhancement of bulk enzyme preparation methods, the acquisition of enzymes with superior catalytic properties, and the development of sophisticated approaches for pathway design and system optimization. In alignment with the workflow of ivBT development, this chapter presents a systematic introduction to pathway design, enzyme mining and engineering, system construction, and system optimization. The chapter also proffers perspectives on ivBT development., (© 2023. The Author(s), under exclusive license to Springer Nature Switzerland AG.)