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This group-randomized trial assessed the effects of a universal prevention training curriculum for school administrators and teachers that focused on effective strategies to prevent adolescent substance use and related problems. Twenty-eight schools in three regions of Peru were randomly assigned to either an intervention or control condition (14 schools per condition). Repeated cross-sectional samples of 11 to 19-year-old students participated in four surveys from May 2018 to November 2019 (N = 24,529). School administrators and teachers at intervention schools participated in a universal prevention training curriculum focusing on the development of a positive school climate as well as effective policies related to school substance use. All intervention and control schools were offered Unplugged, a classroom-based substance use prevention curriculum. Outcome measures included: lifetime drug use; past-year and past-month tobacco, alcohol, marijuana, and other drug use; awareness of school tobacco and alcohol use policies; perceived enforcement of school policies; school bonding; perceived friends' use of tobacco, alcohol, marijuana and other drugs; and personal problems in general and problems related to substance use. Multi-level analyses indicated significant reductions in past-year and past-month smoking, friends' substance use, and problems related to substance use and in general at intervention relative to control schools. Significant increases were found in intervention vs. control schools related to students' awareness of school substance use policies, perceived likelihood of getting caught for smoking, and school bonding. These findings suggest that the universal prevention training curriculum and the school policy and climate changes it promoted reduced substance use and related problems in the study population of Peruvian adolescents.