Background In-depth studies highlight that trust is essential for the effective interaction with autonomous vehicles, which, have not yet gained public trust. Objective The aim of this study was to develop and validate a comprehensive questionnaire to evaluate people's trust in autonomous vehicles. Methods After identifying influential factors in trust by interviews and brainstorming sessions, 75 questions across 5 dimensions were developed, which were then narrowed to 69 in 3 dimensions using “thinking aloud”. This version was assessed by 24 experts, resulting in 19 responses and the exclusion of 22 questions based on CVI and CVR. Finally, the reliability was determined using Cronbach's Alpha after an experiment with 24 participants. Results A 47-item questionnaire, with 3 dimensions including personal, social, and technical factors, and 21 sub-dimensions was developed. The lowest CVI (0.63) was for “mental complexity” and the highest (0.90) belonged to “personality”. The least CVR (0.56) for “meaningfulness attitude” was acquired, while the highest CVR (1.00) was recorded for 9 questions. Total Cronbach's Alpha coefficient (0.93) showed satisfactory reliability. Conclusions This validated instrument offers a comprehensive tool for evaluating trust in AVs for future studies.