This study's goal was to establish the prevalence of driving under the influence of alcohol (DUI) and alcohol consumption patterns among drivers in Cali, Colombia, in 2013. A cross-sectional study based on a roadside survey using a stratified and multi-stage sampling design was developed. Thirty-two sites were chosen randomly for the selection of drivers who were then tested for blood alcohol concentration (BAC) and asked to participate in the survey. The prevalence of DUI was 0.88% (95% confidence intervals [95% CI] 0.26%–1.49%) with a lower prevalence when BAC was increasing. In addition, a higher prevalence was found during non-typical checkpoint hours (1.28, 95% CI −0.001%–0.03%). The overall prevalence is considered high, given the low alcohol consumption and vehicles per capita. Prevention measures are needed to reduce DUI during non-typical checkpoints and ongoing studies are required to monitor the trends and enable the assessment of interventions.