Objectives: Rat models have been successfully used to study dental fluorosis, but the quantification of enamel alterations is difficult since conventional methods to evaluate mineral density (hardness, microradiography, polarized light) can only detect alterations caused by high fluoride concentrations (e.g. >50 μg F/mL in the water). Yet, rat incisors present an orange color due to iron pigmentation, but upon exposure to fluoride, even at concentrations as low as 10 μg F/mL, white striae are observed, and their intensity increases with F exposure. Thus, the aim of this study was to develop a sensitive index to quantify fluorosis in rat incisors based on the analysis of the intensity of the orange and white striae. Methods: Sixteen 35-days-old Wistar female rats received water containing 0, 12.5, 25 or 37.5 g F/mL. After 78 days, enough time to allow the full growth of the incisors, animals were sacrificed, and F concentrations in plasma and femur were determined. The Dental Fluorosis by Image Analysis (DFIA) index, based on the difference of the average pixel intensity of the orange (higher intensity) and white (lower intensity) striae caused by fluoride, was used to quantify fluorosis in grayscale cuts (690x170 pixels) from digital images taken from the rats manbible incisors. Results: A significant linear increase in F concentration in plasma and bone was observed according to the increase in F concentration in the water (r=0.82 and 0.98, respectively, p<0.0001). Fluorosis index for groups 0, 12.5, 25, 37.5 ug F/mL was, respectively, (meanSD, n=4) 1.10.3, 1.70.6, 2.90.7 and 4.10.9, with a significant linear correlation being found (r=0.85, p<0.0001). Conclusion: The results suggest that the developed DFIA index is a simple and valid method that can be successfully used to quantify mild alterations induced by fluoride in rat incisors.