Pharmacists are increasingly involved in strategies to fight antimicrobial resistance by ensuring optimised antibiotic (AB) use, including adherence support. Successful adherence interventions should be tailored to patients' barriers and validated instruments are needed. This study aimed to identify adherence barriers to AB treatment, develop a self-report questionnaire, and validate it in outpatients.; Adherence barriers were identified through a systematic literature search and focus group discussion. Unmodifiable and irrelevant barriers were excluded from further processing. A validation study assessed the questionnaire's internal reliability and construct validity by comparing the questionnaire's score with electronically monitored adherence data.; A 15-item questionnaire was created. Overall, 68 patients were included in the construct validation analysis (60.3% female). The mean consecutive taking adherence was 88% and the most frequently reported barriers were "worries about side-effects" (37%) and "having swallowing difficulties" (19%). Three items were excluded from the questionnaire, which was supported by an increase of Cronbach's alpha (0.69 to 0.70). The 12-item version's score correlated significantly with medication adherence rate (r = -0.34,; p; < 0.01).; The self-report questionnaire is a reliable and valid tool to pre-emptively assess adherence barriers in outpatients prescribed ABs. In the future, appropriate adherence interventions can be matched to barriers and tested in a pilot intervention study.