Integrated bio-refineries offer a potential for a more sustainable production of fuels and chemicals. However, the sustainability implications of integrated bio-refineries are still poorly understood. Therefore, this work aims to contribute towards a better understanding of the sustainability of these systems. For these purposes, a methodological framework has been developed to assess the sustainability of different 2nd generation feedstocks to produce bio-ethanol, energy, and platform chemicals using bio-chemical or thermo-chemical routes in an integrated bio-refinery.The methodology involves environmental, techno-economic, and social assessment of the bio-refinery supply chain. Life cycle assessment (LCA) is used for the environmental assessment. The economic assessment is carried out using life cycle costing (LCC) along side traditional economic indicators such as net present value and payback period. Social issues such as employment provision and health and safety are considered within the social sustainability assessment. The methodology has been applied to two case studies using the bio-chemical and the thermo-chemical conversion routes and four feedstocks: wheat straw, poplar, miscanthus and forest residue.For the conditions assumed in this work and per litre of ethanol produced, the LCA results indicate that the thermo-chemical conversion is more environmentally sustainable than the bio-chemical route for eight out of 11 environmental impacts considered. The LCA results also indicate that the main hot spot in the supply chain for both conversion routes is feedstock cultivation. The thermo-chemical route is economically more sustainable than the bio-chemical because of the lower capital and operating costs. From the social sustainability point of view, the results suggest that provision of employment would be higher in the bio-chemical route but so would the health and safety risks.