The grafting of the rootstock with the lemon tree is an agronomical technique used to improve production and/or quality of the fruit. The interstock has been used with different fruit trees to modulate the tree size, fruit production and quality, and the aging of the tree. The lemon trees grafted with interstocks increase their longevity, lemon production and quality; interstocks are also used to decrease the thickness of the trunk at the grafting point. This enlarging of the trunk provokes a decrease of the sap flow. In our study, "Verna" lemon trees were grafted with interstock between the rootstock and the lemon tree to follow the flavonoid content of the lemon juice. The lemon juice was obtained from the lemons collected of the grafted lemon trees. Two types of rootstocks were used: Citrus aurantium L. and Citrus macrophylla L. Seven interstocks from five cultivars of orange tree, one cultivar of lime tree, and one cultivar of tangerine tree were used. "Verna" lemon trees were also grafted directly to the rootstock. The rootstock was more important agronomic factor than the interstock on the total flavonoid content of lemon juice. The interstock grafting had only a small influence on the flavonoid content of the lemon juice, and it modulated the individual flavonoid content. Citrus aurantium L. rootstock and "Berna" and "Washington Navel" interstocks were the most appropriate to graft in the lemon tree. This interstock grafting technique does not increase the flavonoid content of the lemon juice. Regarding the individual flavonoids, the 6,8-di-C-glucosyl diosmetin was the most affected flavonoid by the type of rootstock used. The interstock used is able to alter the individual quantitative flavonoid order of eriocitrin, diosmin, and hesperidin. In addition, the HPLC-ESI/MS(n) analyses provided the identification of two new flavonoids in the lemon juice: Quercetin 3-O-rutinoside-7-O-glucoside and chrysoeriol 6,8-di-C-glucoside (stellarin-2). The occurrence of apigenin 6,8-di-C-glucoside (vicenin-2), eriodictyol 7-O-rutinoside, 6,8-di-C-glucosyl diosmetin, hesperetin 7-O-rutinoside, homoeriodictyol 7-O-rutinoside and diosmetin 7-O-rutinoside was also confirmed in lemon juice by this technique.