Due to permanent contact with bodily secretions such as blood and saliva, the dental workplace poses a high risk of infection for patients as well as for personnel. High-speed dental instruments are still considered one of the major hygienic risks, as the high-speed rotation of the attachments leads to the retraction of infectious material from patients' oral cavities. The aim of this study was to investigate the extent to which dental handpieces are contaminated after use. Spray-water samples were taken from different handpieces used in seven dental offices and protein concentrations were measured photometrically. In the first part of the study, samples were collected from each handpiece before and after the treatment of the patients. Additionally, the changes in protein concentration after consecutive treatments in which the same high-speed dental instrument was used were investigated. The results demonstrated measurable protein concentrations in 91.2% of a total of 398 samples, and 96.4% of the spray-water samples taken after treatment showed a discrepancy from the initial measured protein concentration. In 68.4% an increase in protein concentration was observed, whereas in 27.9% a decrease was measured. In conclusion, the internal contamination of high-speed dental instruments frequently occurs in daily usage and consequently may lead to the transmission of infectious agents by flushing the contaminated water out of the spray water tubes. Moreover, it must be pointed out that internal cleansing of handpieces is insufficient and that a final mechanical disinfection is indispensable.