Recent advances in synthetic biology and metabolic engineering have enabled the production of a range of chemicals using engineered microorganisms1,2. However, despite the intriguing potential, the biological production of high-value chemicals is challenging because it is likely to have low titer and substantial amounts of excreted byproducts, and the purity specifications for high-value chemicals, as opposed to fuels, are rather strict, which means that separation costs are likely to represent a large fraction of the total production cost (more than 70% of total cost3). Thus, the efficient synthesis of bio-separation processes becomes a critical task. Although this synthesis problem has been studied for various chemicals in the past, these studies were mostly performed on a case-by-case basis. There has been limited research towards the development of systematic methods for bio-separations, applicable to all chemical targets. Accordingly, the goal of the present work is to develop a general bio-separation superstructure optimization framework, aiming to provide guidance on the preliminary synthesis of separation networks.