Background: Organisational culture (OC) has increasingly become a crucial factor in defining healthcare practice and management. However, there has been little research validating and adapting OCAI (organisational culture assessment instrument) to assess OC in healthcare settings in developing countries, including Vietnam. The purpose of this study is to validate the OCAI in a hospital setting using key psychometric tests and confirmatory factor analysis (CFA). Methods: This is a cross-sectional study. Self-administered structured questionnaire was completed by 566 health professionals from a Vietnamese national general hospital, the General Hospital of Quang Nam province. The psychometric tests and CFA were utilized to detect internal reliability and construct validity of the instrument. Results: The Cronbach's alpha coefficients (α-reliability statistic) ranged from 0.6 to 0.8. In current culture, the coefficient was 0.80 for clan and 0.60 for adhocracy, hierarchy and market dimension, while in expected culture, the coefficient for clan, adhocracy, hierarchy, and market dimension was 0.70, 0.70, 0.70 and 0.60, respectively. The CFA indicated that most factor loading coefficients were of moderate values ranging from 0.30 to 0.60 in both current and expected culture model. These models are of marginal good fit. Conclusions: The study findings suggest that the OCAI be of fairly good reliability and construct validity in measuring four types of organisational culture in healthcare setting in resource-constrained countries such as Vietnam. This result is a first step towards developing a valid Vietnamese version of the OCAI which can also provide a strong case for future research in the field of measuring and managing organisational culture.