Suspected clusters of non-infectious diseases (cancers, degenerative diseases, chronic inflammatory diseases, congenital malformations, etc.) are regularly reported to regional health agencies (ARS), raising questions about risk factors present in the various living environments and surroundings of the individuals concerned. These are warning signs that must be evaluated by public health authorities and agencies as part of their missions to monitor and protect public health against risks. Following an initial guide published in 2005, this new edition incorporates the experience gained since then, updates to knowledge and guidelines produced in other countries, methodological developments, and the need to better account for and manage the local, social, and media context. The guide provides a reference framework for public health professionals in public health agencies, Regional Health Agencies (ARS), and researchers involved. A spatio-temporal cluster is an “unusual grouping in time and space, whether real or perceived, of health-related incidents.” The investigation is based on a progressive and prioritized collection of information with milestones to assess the relevance of whether or not to initiate new investigations or actions. While the limitations of these retrospective investigations restrict the ability to explain the excess of cases for many clusters, in certain situations, the investigation can provide new insights to control exposure to risk. The step-by-step approach aims to identify situations in which an etiological study is most likely to be conclusive. The guide is structured in four steps: 1: handling the report; 2: analysis and validation of the cluster; 3: necessity and feasibility of a specific epidemiological study; 4: implementation of a specific etiological epidemiological study if decided in step 3. Since reports of suspected clusters often occur in specific local and social contexts, it is essential to analyze them to understand them and adapt the public health response. Each step must ensure active listening, information sharing, dialogue, and regular consultation with patients, the public, their representatives, and stakeholders. The decision to continue or halt the investigation at each stage is based on an analysis of the following criteria: whether the initial objectives of the investigation have been met; the specificity of the health event; the usual frequency of the health event; severity; the magnitude of the excess cases; the vulnerability of the population (children, newborns, etc.); the existence of exposure to a risk factor shared by a sufficient number of cases and capable of explaining the excess; the state of knowledge regarding the links between exposures and the disease; the scientific plausibility of the link between exposure and the disease; the potential for further development; the context and social expectations; the need for management measures to reduce potential risk exposure. These principles apply to all clusters regardless of the disease and the population concerned. There are specific considerations for pediatric cancers, congenital malformations, and those occurring in the workplace. Five appendices supplement the guide on specific aspects (pediatric cancers, congenital malformations, and occupational clusters), statistical approaches, the assessment of the social context, and finally the consideration of ethical dimensions and personal data protection.