Background: Previous research suggests that smoking cessation interventions are poorly implemented. This study reports the development and testing of a questionnaire including knowledge, attitude, behavioral, and organizational (KABO) factors affecting the implementation of smoking cessation practices in hospitals by health care providers and organizations. Methods: An initial pool of 44 items was developed to assess the individual knowledge, attitudes, and beliefs of health professionals towards smoking cessation practices according to the 5 A's intervention model, as well as organizational barriers and opportunities for its implementation. Items were measured in a scale from 0=“Not at all/Never” to 10 = “Completely/Always”. Data were collected from health workers (n = 702) in Catalonia. The validity of the instrument was measured by: (a) analyzing the items, (b) assessing the internal structure, (c) estimating the internal consistency, and (d) analyzing the relationship between this tool and the 5 A's intervention model. Results: Seven domains were extracted: individual skills, positive organizational support, attitudes and beliefs, individual commitment, organizational resources, beliefs about patient desire/readiness to quit, and organizational endorsement. These domains explained 69.7% of the variance, and allowed for the development of a refined 26-item version of the questionnaire. Both the seven domains and the total scale showed adequate internal consistency. Conclusions: Psychometric testing indicates that the KABO questionnaire is a reliable and valid instrument for assessing the main barriers and facilitators to smoking cessation intervention implementation. Individual factors better explained the implementation of smoking cessation interventions in hospitals, and the seven identified domains can be used for further investigations into how the implementation of evidence-based practices impacts smoking cessation performance.