Healthcare-Associated Infections present a major threat to patient safety globally. According to studies, more than 50% of HAI could be prevented by proper hand hygiene. Effectiveness of hand hygiene is regularly evaluated with the fluorescent method: performing hand hygiene with a handrub containing an ultra violet (UV) fluorescent marker. Typically, human experts evaluate the hands under UV-A light, and decide whether the applied handrub covered the whole hand surface. The aim of this study was to investigate how different experts judge the same UV-pattern, and compare that to microbiology for objective validation. Hands of volunteer participants were contaminated with high concentration of a Staphylococcus epidermidis suspension. Hands were incompletely disinfected with UV-labeled handrub. Four different UV-box type devices were used to take CCD pictures of the hands under UV light. Size of inadequately disinfected areas on the hands were determined in two different ways. First, based on microbiology; the areas where colonies were grown were measured. Second, four independent senior infection control specialists were asked to mark the missed areas on printed image, captured under UV light. 8 hands of healthy volunteers were examined. Expert evaluations were highly uncorrelated (regarding interrater reliability) and inconsistent. Microbiology results weakly correlated with the expert evaluations. In half of the cases, there were more than 10% difference in the size of properly disinfected area, as measured by microbiology versus human experts. Considering the result of the expert evaluations, variability was disconcertingly high. Evaluating the fluorescent method is challenging, even for highly experienced professionals. A patient safety quality assurance system cannot be built on these data quality.