A novel framework for assessing the sustainability of wild species use is presented to address weaknesses in current formulations and support global and national policies relating to sustainable use. The novelty of the framework is its addition of animal health and welfare and human health to the conventional ecological, social, and economic dimensions of sustainability. The five-dimensional sustainability assessment framework (5DSAF) consists of 42 principles, which have been derived from an analysis and synthesis of existing international, national, sectoral and species-specific guidelines and standards. It can be applied by use of an Excel-based tool that allows scores to be allocated to each principle with results graphically displayed in the form of a radar chart. The 5DSAF has been successfully piloted as a self-assessment tool at industry and enterprise level and has the potential to evolve into a universal standard that government, private sector, and civil society actors could use to assess the sustainability, legality, and safety of all value chains for wild species and products.