Aquaculture is one of the fastest-growing food-producing sectors worldwide, making it desirable to assess the sustainability of aquaculture systems. The objective of this study was to develop a portfolio of quantitative indicators of economic, environmental and social sustainability to assess different aquaculture systems. The indicators were developed from 2003 to 2016, combining top-down and bottom-up methods, together with practical observations in experimental and commercial aquaculture facilities. A total of 56 economic (14), environmental (22) and social (20) indicators are proposed. Economic sustainability indicators reveal the degree of efficiency in using financial resources, the economic feasibility, resilience, and the capacity to absorb negative external costs and to generate funds for reinvestment. Environmental indicators reflect the use of natural resources, the efficiency in using resources, the release of pollutants and unused byproducts, and the risk of reducing biodiversity. Social sustainability indicators reflect the capacity to generate benefits for local communities, including jobs and food security, equitable income distribution, equality of opportunity, and inclusion of vulnerable populations. The indicators thus developed can be used on farm, regional, global or sectorial scales. They are quantitative, broad, scientifically sound, easy to understand and interpret, feasible to obtain on farms or on research stations, and permit comparison at different scales of space and time. Thus, they can be used to assess production systems and to compare different experimental treatments in research experiments. They also can be used by certifying organizations, investors, and policymakers. They allow performing diagnostics, identifying strengths and weaknesses, setting goals and determining actions, and assessing the effectiveness of actions and public policies.