The number of patients in need of life-saving organ transplants continues to exceed the number of available. One of the most important factors influencing peoples’ practices and rates of organ donation is their attitude and beliefs toward it. However, there is no validated comprehensive instrument that evaluates peoples’ attitudes toward organ donation. Thus, this study aimed to develop and validate such tool. A questionnaire was prepared after reviewing literature. The initial items (n = 45) were evaluated by 15 nonexperts pilot to verify face validity, then content validity was established by three experts, and construct validity was established by Exploratory Factorial Analysis. Afterward, internal consistency was checked using Cronbach’s alpha analysis. Eventually, a total of 31 items were included in the questionnaire, distributed on four subscales. According to the results, we found that the Public Attitude toward Organ Donation questionnaire has adequate validity and reliability to assess the peoples’ attitudes toward organ donation.