Background Cultural competence of healthcare professionals (HCPs) is recognized as a strategy to reduce cultural disparities in healthcare. However, standardised, valid and reliable instruments to assess HCPs' cultural competence are notably lacking. The present study aims to 1) identify the core components of cultural competence from a healthcare perspective, 2) to develop a self-report instrument to assess cultural competence of HCPs and 3) to evaluate the psychometric properties of the new instrument. Methods The conceptual model and initial item pool, which were applied to the cross-cultural competence instrument for the healthcare profession (CCCHP), were derived from an expert survey (n = 23), interviews with HCPs (n = 12), and a broad narrative review on assessment instruments and conceptual models of cultural competence. The item pool was reduced systematically, which resulted in a 59-item instrument. A sample of 336 psychologists, in advanced psychotherapeutic training, and 409 medical students participated, in order to evaluate the construct validity and reliability of the CCCHP. Results Construct validity was supported by principal component analysis, which led to a 32-item six-component solution with 50% of the total variance explained. The different dimensions of HCPs' cultural competence are: Cross-Cultural Motivation/Curiosity, Cross-Cultural Attitudes, Cross-Cultural Skills, Cross-Cultural Knowledge/Awareness and Cross-Cultural Emotions/Empathy. For the total instrument, the internal consistency reliability was .87 and the dimension's Cronbach's alpha ranged from .54 to .84. The discriminating power of the CCCHP was indicated by statistically significant mean differences in CCCHP subscale scores between predefined groups. Conclusions The 32-item CCCHP exhibits acceptable psychometric properties, particularly content and construct validity to examine HCPs' cultural competence. The CCCHP with its five dimensions offers a comprehensive assessment of HCPs' cultural competence, and has the ability to distinguish between groups that are expected to differ in cultural competence. This instrument can foster professional development through systematic self-assessment and thus contributes to improve the quality of patient care.