Consideration of alternative transport fuels, produced from feedstocks and renewable resources, is inevitable to enhance energy security and alleviate environmental burdens. Whilst these fuels are apparently considered to be clean, they may not be entirely sustainable from economic, environmental and social perspectives. A Life Cycle Sustainability Assessment (LCSA) framework that integrates all three components of the Triple Bottom Line (TBL) sustainability can potentially be used to evaluate the sustainability performance of fuels from well to wheel. This paper presents an LCSA framework consisting of Environmental Life Cycle Assessment (ELCA), Life Cycle Cost (LCC) and Social Life Cycle Assessment (SLCA) tools to assess the environmental, economic and social performance of alternative fuels. The framework is aimed at identifying the areas that require improvements for overall sustainability performance. The proposed framework provides a comprehensive basis that considers the region-specific variations in the life cycle data pertaining to alternative fuels. The added feature of the framework is its robustness to accommodate variations in natural resources, and other regional issues, such as socio-economic and demographic changes. The framework has been tested using a hypothetical example of canola-based biodiesel.