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Research on adolescent substance use is of utmost importance. Using local toxicological data, both prevalence and pattern of substance use (SU) and substance-related death (SRD) can be assessed to design effective prevention programs. A retrospective study of toxicology investigations of all adolescents referred to the medico-legal section of the Toxicology Unit of the University Hospital of Lille, France, for a 2-year period from 2017 to 2018. In the total sample of 1961 cases, adolescents accounted for 3.3% of the cases (n = 65). Among the adolescents, 16.9% were aged 10–14 years and 83.1% were aged >14–19 years. About 69.2% were males. Less than 70% of all presented adolescents used substances. More than two-thirds (74%) of positive detections were male. Illicit substances (43%) were the most detected substance followed by alcohol (20%) and prescription substances (20%). Tetrahydrocannabinol (THC) was extremely common as it was found in 29% of all adolescents. Cocaine and amphetamines were detected in 13.8% of total tested adolescents. Polysubstance use was common between alcohol and THC and among males. About one-third of deaths were due to substance use. About 54% of SRD was associated with polysubstance detection. It is recommended that illicit substances, ethanol, and prescription substances are targeted for testing among adolescents in order to provide appropriate prevention.