Compliance with hand hygiene is important in infection control, and the rate of hand disinfectant application is rising in recent years. Therefore, we evaluated the qualitative evaluation system of hand hygiene by mechanically measuring hand disinfectants containing a fluorescence substance. We observed that the hospital staff and general population had a significantly higher application rate of hand disinfectant on the palm side than on the dorsal side (P < 0.05). Hospital staff had a significantly higher application rate compared to the general population (P < 0.05). Disinfectants on the bad hand hygiene area are distributed more on the dorsal side than on the palm side in both hospital staff and general population. Particularly, the bad hand hygiene area was around the thumb of both hands. Disinfectant distribution on the bad hand hygiene area was evaluated by GlitterBug, particularly on the fingertips, which was different from the evaluation using HandInScan. It was estimated that disinfectant distribution on the bad hand hygiene area is associated with hand hygiene techniques, amount of hand disinfectant applied, and other factors. Therefore, evaluation and appropriate instruction on hand hygiene methods is necessary. HandInScan and GlitterBug both had advantages, therefore it seemed useful to choose them as appropriate. In addition, the involvement of pharmacists in the evaluation of such systems is meaningful for contributing to more effective and appropriate use of hand disinfectants.