Multiple stressors resulting from anthropogenic actions are increasingly recognized as a major threat to aquatic ecosystems. In general, ecosystems change as they respond to multiple threats that interact in complex ways, depending on the natural environment. This scenario poses complex tasks for researchers, managers, and policymakers, which require a well-defined framework to provide a clear roadmap that identify steps to apply in adaptive management decisions, monitoring designs and policy implementation. Building upon core elements of previous work, we present a Multiple Stressors Assessment Framework (MSAF) for aquatic ecosystems that provides a roadmap on the achievement of an improved integration between monitoring designs, data acquisition, evaluation processes, and management actions. The MSAF involves seven steps: 1) problem formulation and the definition of the ecosystem type and the spatial-temporal scale, 2) data compilation on the ecosystems' environmental characteristics and the definition of the type, identity and intensity of environmental stressors, 3) data compilation/collection on the biological/ecological receptors (endpoints) to stressors and selection of response variable to monitor (based on structure-based indicators and functional metrics), 4) characterization of the stressor-response relationships and their interactions, 5) construction of specific ecological conceptual models and choice of adequate statistical approaches to test the conceptual models' viability, 6) generation of hypotheses on interactive effects on biological/ecological endpoints, validation of models, hypotheses testing and, if possible, comparison of results with controlled experiments in realistic settings, and 7) recommendations to adaptive monitoring and if necessary, improvement of the study design, and eventual inclusion of other hypotheses and statistical approaches in the context of adaptive management actions. We review progress made in Europe, the USA and Canada in this field using case examples, highlight the approaches taken by the different jurisdictions that align with our framework and identify the linkages between multiple stressors assessments and decision-making for each region. We conclude that a disconnection remains between the investigation of the combined effects of multiple stressors and the implementation of management practices and policy translation. The way forward is through a collaborative effort to create standardized methodologies and appropriate programmes in this field. We hope the proposed framework can be used as a foundation to diagnose multiple stressor interactions and identify responses of ecological indicators to inform effective adaptive management of freshwater ecosystems globally