Objective: To identify and compare tools to assess Evidence-Based Practice (EBP) behaviour among healthcare professionals. Design: Systematic review. Data sources: MEDLINE, EMBASE, Cochrane Library, PsychInfo and CINAHL up to July 2011. Study selection: Titles, abstracts and eligible full text articles were screened by two reviewers independently. Data extraction: Relevant data were extracted by one reviewer and checked by a second reviewer. Eligibility criteria for selecting studies: original studies among all healthcare professionals that described the development or use of EBP behaviour assessment tools. Results: Of 19 310 identified articles, 172 studies were included. We identified 117 questionnaires, 10 interviews or focus groups, nine observational studies, 27 chart evaluations and nine studies used a combination of methods. Psychometric properties of the questionnaires used were reported in about half of the studies, in seven studies that assess a single EBM step and in six studies that assess a combination of EBM steps. One of these assessed all five steps of EBP. Conclusions: Valid and reliable EBP behaviour assessment tools are available. However, only one questionnaire validly assessed all five EBP steps, covering the entire concept of EBP.