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Few studies have investigated deployment-related experiences of healthcare workers dispatched for medical humanitarian aid or attempted to assess their difficult living and working environments. This is the first study to develop and validate a scale to measure these kinds of difficulties, in 264 Japanese healthcare workers. The Humanitarian Aid Difficulty Scale was developed in three stages. First, an item pool was generated based on literature and expert reviews. The scale was then tested in a pilot study. Reliability and validity were identified through exploratory and confirmatory factor analysis and Cronbach's alpha. The scale consisted of 23 items across five factors based on exploratory factor analysis (cooperation, health status, infrastructure, culture and customs, and supplies and equipment). The total variance explained was 60.7%. Reliability of the five factors was acceptable and validity was supported by confirmatory factor analysis. Cronbach's alpha for the scale was 0.87. The scale may enable evaluation of the level of difficulty of the living and working environments of Japanese healthcare workers in medical humanitarian aid who are at a greater risk of distress.