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The environmental persistence of a substance plays a key role in determining its exposure to humans and other organisms, making this an important component in the risk assessment and management of chemicals. Regulatory persistence assessments generally involve a comparison of degradation half-lives against threshold criteria for different environmental compartments, typically water, sediment, and soil. Half-lives are commonly determined using Organisation for Economic Cooperation and Development (OECD) guideline biodegradation simulation tests. Other information may be considered relevant to persistence assessments, such as results from biodegradation screening tests, quantitative structure-activity relationships, field studies, monitoring data, and non-standard laboratory experiments. All available relevant information should be considered together in a weight-of-evidence approach, but clear guidance is currently lacking both for evaluating the quality of individual studies and for combining these in a single weight-of-evidence determination. Here we present a systematic methodology to collate, evaluate, and integrate relevant information to reach robust, transparent and consistent conclusions for persistence assessments. First, the quality (reliability and relevance) of individual studies within each information category, or ‘line of evidence’, is evaluated using a novel scoring methodology. Then, information from different studies is combined to determine outcomes for each line of evidence. Finally, a stepwise weight-of-evidence approach is applied to integrate outcomes from different lines of evidence to reach an overall conclusion for the persistence assessment. Consistency of information is evaluated at various stages in line with weight-of-evidence best practice. The methodology has been developed in accordance with principles of the European Union (EU) Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) regulatory framework, test guidelines and guidance, whilst being flexible to accommodate different regulatory practices. The methodology has been implemented in a freely available Excel-based software tool, the Persistence Assessment Tool, and is demonstrated using a case study substance hexabromocyclododecane.