The aim of this study was to objectively assess the hand hygiene performance of medical students. Hand rubbing technique was evaluated by employing innovative UV-light-based imaging technology, identifying patterns and trends in missed areas after applying WHO's six-step protocol. This specially designed hand hygiene education and assessment program targeted 1,344 medical students at two distant sites in Central Europe. Students were introduced to a short video, presenting the basics of hand hygiene, and then received further demonstration from professional trainers, focusing on the correct execution of WHO's six-step technique. To verify the acquired skill, participants rubbed their hands with UV-marked alcohol-based solution. Digital images of the hands were recorded under UV light, followed by computer evaluation and assessment. Immediate objective visual feedback was given to the participants showing missed areas on their hands. The statistical analysis of missed spots was based on retrospective expert-driven manual evaluation. Significant difference in rubbing quality was found between female and male participants [35.3% (CI 95%: 33-38%) versus 29.0% (CI 95%: 27-31%), p < 0.001], dominant and non-dominant hands [43.4% (CI 95%: 39-48%) versus 34.9% (CI 95%: 32-38%), p = 0.002], and various zones of the hands' dorsal side. Based on the participants' feedback and the evaluation of the infection control specialists, it can be stated that the identification of typically missed patterns and the instant visual feedback have a vital role in improving the hand hygiene technique of prospective medical staff. Keywords: hand hygiene assessment, fluorescein trial, medical students training Introduction "Have we forgotten the lesson of Semmelweis?" -stands the question in Lancet [1], referring to the generally weak hand hygiene (HH) performance of physicians in hospitals. Fortunately, the 4% compliance rate reported at a gynecology ward represents an extremely low value [2], but the usually reported rates of 20-50% are still far below expectations [3][4][5]. The main reported reasons for this poor performance include the tight time schedules, forgetfulness, unsatisfactory knowledge on HH guidelines, denying the importance of hands as a source of infection, negative influence of older, more experienced specialists, and skin irritations caused by hand rub solutions [6,7]. Education and training play a vital role in promoting HH [8,9].It is a widely shared concept that the nursing staff shows a better compliance on average than the physicians [3,10]. The unsatisfactory quality of knowledge is clearly supported by the observation that according to physicians' opinion, less HH moments are enough than the amount believed necessary by other healthcare workers [11]. As an interesting analogy, the question of antibiotic resistance is generally believed of high importance by medical students; however, it will be treated superficially during their later work as physicians, similarly to the question of HH [12]. Nurses consider the d...