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The recreational use of nitrous oxide (N2O) is an emerging public health issue. Chronic N2O abuse may result in various clinical symptoms, and involve neurological, psychiatric, and cardiovascular outcomes. Despite the difficulties involved in the laboratory investigation of N2O intoxication, there are currently no guidelines in France to help both clinicians and biologists use appropriate biomarkers for the diagnosis and monitoring of patients with clinical symptoms potentially related to N2O intoxication. A multi-disciplinary working group, organized under the auspices of the Société Française de Biologie Clinique (SFBC) and in collaboration with the Sociétés Françaises de Médecine d’Urgence (SFMU), Toxicologie Analytique (SFTA), Thrombose et d’Hémostase (SFTH), Vitamines et Biofacteurs (SFVB), and the Fédération Française de Neurologie (FFN), was recently implemented to develop practical guidelines. The methodology of the working group is based on the critical analysis of the literature, and concerns and objectives are grouped into five working packages. This article aims primarily to expound upon the methodology and objectives of the ongoing SFBC Working Group on N2O.