The sustainability of biofuel operations encompasses several issues which are key to promoting uptake in a global environment. However, much of the analysis of biofuel production tends to be dominated by life cycle assessment (LCA) for assessing environmental burdens, coupled with techno-economic assessment. Whilst LCA is robust, using it in holistic sustainability assessment alone would be of limited value. This chapter provides a broad discussion of salient sustainability aspects of biofuel production. A discussion of these aspects shows that it is necessary to go beyond LCA in order to effectively characterize the sustainability of bio-products. In addition, multi-criteria decision analysis is discussed as sustainability assessment lends itself well to these methods in order to promote sustainability strength and decision power. Finally, a broad, inclusive assessment framework for biorefineries is proposed that promotes accountability, embracing uncertainty and adherence to sustainability guiding principles and frameworks.