This study aimed to refine and validate a Mechatronic System Usability Evaluation (MSUE) questionnaire. A total of 626 users were selected using random sampling, from the area of West Thessaloniki, Greece. The validity of the questionnaire were tested with the content and construction validity method. The reliability of the MSUE questionnaire instrument were tested using test-retest and internal consistency method. Factor analysis resulted a 25 questions questionnaire divided into five axes of, namely efficiency, effectiveness, Satisfaction, ease of use, and ease of learn. The Cronbach Alpha coefficient for the entire scale was 0.819. The questionnaire was tested and the results was shown a suitable instrument to measure usability on mechatronic systems.