The influence of rootstock, maturity and extraction settings on limonin, hesperidin and narirutin levels of orange juice was evaluated. A liquid chromatographic method to determine limonin was developed and validated. The linear range was 0.410 to 61.5 µg.mL-1, with a linear correlation coefficient higher than 0.999. The limit of detection was 0.144 µg.mL-1and limit of quantification 0.363 µg; precision showed RSD≤5.0% and accuracy was from 92.6 to 100.4%. Limonin was identified in Pêra-Rio orange juices from Cleopatra mandarin and Rangpur lime rootstocks extracted in the NFC and FCOJ settings during the 2013 harvest. Limonin levels in Pêra-Rio orange juices ranged from 0.86 to 3.94 µg.mL-1 and flavonoids hesperidin and narirutin levels ranged from 12.00 to 26.02 µg.mL-1 for narirutin and from 122.12 to 175.01 µg.mL-1 for hesperidin. Principal component analysis was able to differentiate the juices from Cleopatra mandarin and Rangpur lime rootstocks according to ripeness, as well as extraction settings. Limonin and flavonoid levels reduced during maturation. Limonin levels were more expressive at the beginning of the harvest, especially in juice obtained with the FCOJ extraction setting. Hesperidin levels were about ten times higher than narirutin levels in all juices.