Objective: Rat models have been successfully used to study enamel defects caused by chronic exposure to fluoride (F), but the quantification of these defects, especially when mild alterations are present, is still a challenge. Also, based on the effect of F changing the iron pigmentation of the rat incisor enamel surface, we have developed a method to quantify dental fluorosis by image analysis. The aim of this study was to compare 3 different methods to evaluate dental fluorosis in rats: tranverse microradiography (TMR), cross-sectional hardness (CSH) and dental fluorosis by image analysis (DFIA). Methods: Thirty-five 35-days-old Wistar female rats received water at increasing F concentrations: 0, 12.5, 25, 50, 62.5, 75 or 100 g F/mL. After 70 days, animals were sacrificed, F concentrations in plasma and femur were determined and dental fluorosis was analyzed on the incisors. The upper incisors were longitudinally sectioned, and TMR and CSH measured from 10 to 70 m from the surface. DFIA measured the difference of the average pixel intensity of the orange (lower intensity) and white (higher intensity) striae from digital images taken from mandibular incisors. Linear regression analysis was used to correlate the administered constant F concentrations in water with the respective measured F concentration in plasma and bones, and dental fluorosis. Results: A significant linear increase in the metabolic variables were observed according to the increase in F concentration in the water (p<0.0001, r=0.89-0.98). TMR (p<0.0001, r=0.95) and CSH (p=0.001, r=0.55) were only able to detect the fluorotic defect in the rat incisor at high F concentrations (≥50 gF/mL). DFIA was able to quantify dental fluorosis, even at low F concentrations (p<0.0001, r=0.77). Conclusions: This study suggests that among the evaluated methods, dental fluorosis by image analysis is the most suitable method to quantify small alterations caused by F on rat enamel.