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Purpose Knowledge advancement exposes the pharmacists to new ethical and professional challenges in providing pharmaceutical care. The pharmacist's attitude towards the ethical challenges of pharmacy practice could be of great help in assessing pharmacist's ethical sensitivity. This study aimed to design a valid and reliable questionnaire for measuring pharmacists' attitudes. Methods The present study is the second phase of a sequential exploratory mixed-method study for designing and psychometric evaluation of the questionnaire. In the first phase the questionnaire was developed using semi-structured interviews followed by content analysis. The psychometric evaluation of the questionnaire was performed examining the validity (face, content, and construct), and reliability (internal consistency and Intraclass Correlation Coefficient and Standard Error of Measurement) in a sample of pharmacists practicing in community pharmacies in Tehran and Tabriz, Iran (n = 504). The construct validity of the questionnaire was determined using exploratory and confirmatory factor analysis. Results The Scale-Content Validity Index/Average (S-CVI/AVE) was calculated at 0.84. The results of exploratory factor analysis supported 22 items in four factors including regulations and rules, professional communications, providing and supplying medicine, and consultation and providing pharmaceutical care that are explained by a total of 49.50% of the variance. Also, confirmatory factor analysis confirmed goodness of fit indices and model fit. Cronbach's alpha Coefficient was 0.919 and ICC was 0.914. Conclusions The psychometric evaluation of the present questionnaire shows a native, valid and reliable instrument to assess pharmacists' attitudes toward ethical challenges which could be a mirror of pharmacists ethical sensitivity. [ABSTRACT FROM AUTHOR]