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This study aimed to develop a questionnaire on the quality of working life among female medical and healthcare professionals and examine its validity and reliability. The questionnaire was developed from an item pool drawing on the literature. The four trait scales included 40 items, covering female-specific stress in continuing a career, stress of lifestyle in maintaining personal values, job satisfaction and social support network. The questionnaire’s validity and reliability were assessed using data from 1,784 female doctors, dentists, and nurses. Validity was examined using exploratory factor analysis on each trait for construct validity, and multitrait scaling analysis for convergent and discriminant validity. Reliability was tested using Cronbach’s alpha for trait subscales and scales. Exploratory factor analysis on each trait was convergent. One trait derived three subscales, and another two. The remaining two traits were convergent for one factor. Multitrait scaling analysis showed that all scales and subscales were independent. The questionnaire was therefore internally consistent and had construct validity. Cronbach’s alpha was 0.85 for the total and between 0.72 and 0.83 for the subscales. These results validate the four-trait combination questionnaire and suggest that it would be suitable for use in future research, perhaps in combination with other existing scales.