Life cycle assessment (LCA) is one of the most suitable tool for a uniform assessment methodology of biofuels’ sustainability. However, there are no binding guidelines for LCA of biofuel systems. Published LCAs use a range of methodologies, different system boundaries, impact categories and functional units, various allocation approaches, and assumptions regarding by- and co-products, as well as different reference systems to which the biofuel system is compared. The European Renewable Energy Directive and the US Renewable Fuel Standard focus on greenhouse gas (GHG) emissions. However, previous LCAs of biofuel systems have shown that a reduction of GHG emissions does not lead automatically to a decrease in other environmental impacts, and might in fact be associated with an increase in impacts such as acidification, eutrophication, and land use change. In order to enable effective comparison of biofuel systems, the authors propose a framework for biofuel LCA. System boundaries should be expanded to include the life cycle of by- and co-products. Results should be reported using more than one functional unit. Burden shifting can be avoided by considering an array of impact categories including global warming potential and energy balance, along with eutrophication and acidification potential, and a land use indicator.