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Demineralization around orthodontic brackets is one of the side-effects of orthodontic treatment. However, simple methods for its early detection, quantification and monitoring are lacking. Therefore, the aim of this investigation was to evaluate red laser-light induced fluorescence for measurement of demineralization around brackets in vitro, and whether an interference by the brackets should be expected. The site of measurement was standardized on 30 extracted human molars with natural demineralization on smooth surfaces. Fluorescence was measured (Diagnodent) on prebonded, etched, bonded with stainless steel brackets, and debonded enamel. We found no statistically significant difference in fluorescence after etching. After bonding, the fluorescence decreased by a median value of 0.5 compared to the prebonded and etched enamel. After debonding, the fluorescence increased by a median value of 0.5 and 1 compared with the prebonded and bonded conditions, respectively. The lack of agreement between prebonded and bonded enamel values (limits +12.4/−12.6) was possibly due to the difficulty in finding the same spot without the bracket (coefficient of repeatability = 9.1–10.8). In vitro, demineralization around brackets can be measured by laser fluorescence, but clinical studies are necessary.