Background: Although public bodies need to know drivers’ perception of road safety, in Latin America there are no valid and reliable instruments that propose an integral dimensionality. The objective of this study was to design and validate a Road Safety Perception Questionnaire (RSPQ). Methodology: The design included a review of the available evidence and expert knowledge to select the dimensional items for the instrument. A pilot test was carried out to determine possible corrections and adjustments to the questionnaire, after which a Confirmatory Factor Analysis was performed on a stratified sample of 736 Ecuadorian drivers to determine its reliability and construct validity. Results: The results suggest that the RSPQ has a clear factorial structure with high factorial weight items and good internal consistency. The results of the 41-item model grouped into six dimensions (human, vehicle, road infrastructure, regulatory framework and intervention measures, socioeconomic and driving precautions) obtained the best adjustment indexes at the absolute, incremental and parsimonious levels. Conclusions: The preliminary RSPQ evidence can be considered a valid and reliable instrument to assess drivers’ perception of road safety.