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Objectives: The working situation of railway traffic control rooms (RTCR) employees makes them susceptible to human error. On the other hand, the occurrence of human error by these staff can lead to catastrophic events. Due to the lack of a standard questionnaire in estimating the probability of monthly human errors in employees of the RTCR, this study designs and validates such a tool. Methods: In this mixed-method research, an initial questionnaire of 67 questions was designed, using the literature review and experts’ experiences. To standardize and validate this questionnaire, the opinions of 15 experts, including university professors and experienced people working in control rooms were used. To determine the validity of the questionnaire, two indexes content validity index and content validity ratio were used. Meanwhile, to check the reliability, the weighted kappa coefficient was used. Results: All the initial questions were accepted and a final questionnaire with 67 questions was compiled in two sections for employees of the central control room and employees working in other RTCR. The content validity index values of the questionnaire were 0.9, 0.9 and 0.92 in terms of simplicity, relevancy and clarity, respectively. The content validity ratio value was 0.87. The reliability of this questionnaire was also confirmed by obtaining 73.71%, 87.14% and 80.31% for the minimum, maximum and average percentage of agreement between the questions, respectively. Discussion: The designed questionnaire can estimate the probability of monthly human errors among RTCR employees and can be used in future studies.