Introduction: Hand hygiene, integrated with the proper use of gloves, represents a fundamental activity for the prevention and control of the risk of infection. Despite the advances, adherence to good practices is a challenge, as it is a complex issue, influenced by institutional, cultural and personal factors. Self-efficacy, a belief that an individual has about his or her ability to successfully perform a given activity, is a strong predictor of health behavior. In Brazil, there is still no evidence of validated instruments that involve the determinants of behavior for hand hygiene practices and the use of gloves concomitantly. Objective: To build and validate an instrument for measuring the self-efficacy of health professionals for the practice of hand hygiene and the use of gloves. Method: Methodological study, which proposed to build and validate the Self-efficacy instrument of health professionals for hand hygiene and the use of gloves (AEPS-HML). After the construction of the instrument, the validation process started through face and content validation (Committee of Judges), pre-test and psychometric validation. The evaluation of the metric properties was carried out through validity (face and content and dimensional construct validities, by known and convergent groups) and reliability (test-retest and internal consistency). The significance level of 0.05 was adopted. Results: Version 1 of the AEPS-HML instrument, containing 28 items, was submitted to face and content validation, with five items being excluded. In the pre-test stage, ten nursing professionals responded to version 2 of the AEPS-HML. In the evaluation of the metric properties of version 3 of the AEPS-HML, 362 nursing professionals participated, working in a university hospital, general, large and public. Most participants were female (293 / 80.9%), with a predominant age range of 30 to 59 years (319 / 88.1%) and mean age of 41.6 years (SD = 7.29). As for professional characterization, 151 (41.7%) participants had specialization and 203 (56.1%) worked as a Nursing Technician. The construct validity by known groups was confirmed by comparing the AEPS-HML self-efficacy averages with the variables sex and unit of action variables. The convergent construct validity showed a weak correlation (r = 0.228) between the scores of the AEPS-HML instrument and the Perceived General Self-Efficacy Scale (EAGP). The reliability of the final version (version 4) of the AEPS-HML was adequate (α = 0.77) and the test-retest reproducibility confirmed the instrument's temporal stability. Conclusions: With this instrument, it will be possible to measure the self-efficacy of health professionals for hand care, with a view to minimizing the risk of microbial transmission due to inadequate hand hygiene practices and the use of gloves. The instrument proved to be valid and reliable to evaluate what is proposed and can be used as a tool in the planning of actions for the prevention and control of infections related to health care.