Communities That Care (CTC) is a prevention system aimed at reducing antisocial behaviors in adolescents. In Colombia, this system has been developed and adapted under the name of Comunidades Que se Cuidan (CQC). Successful implementation of CQC depends on valid associations between measured risk and protective factors (RPFs) for substance use and substance use outcomes. This study assessed these associations using large-scale, school-based surveys of Colombian youth. A cross-sectional analysis was performed. Data from 23 communities in Colombia were collected between 2012 and 2016 from young people (N = 50,946) aged 10 to 19 years. Dichotomous alcohol, cigarette, cannabis, and other illegal drug use outcomes were assessed for past 30-day, past-year, and lifetime use. Logistic regression analyses, adjusting for age, gender, and age by RPF, and gender by RPF interactions, were performed for each RPF. All the associations of the 14 RPF evaluated were statistically significant (p<,001). Regarding observed effect sizes, 3.0% were considered very small (0.70 ≥ OR ≤ 1.43), 51.7% small (0.70 ≥ OR ≥ 1.43), 42.6% medium (0.40 ≥ OR ≥ 2.48) and 7.1% large (0.23 ≥ OR ≥ 4.27). Significant main effects for age and gender, and their interactions with RPFs were found for most RPFs. Findings from this study demonstrate the viability of RPFs for adolescent substance use as focal points for intervention planning, development, and evaluation of community-based prevention systems like CQC that rely on epidemiologic data for local decision making.