Plants of the genus Genista has gained interest due to its high content of alkaloids and flavonoids, especially isoflavones, because of their phytoestrogenic activity. Genista monspessulana (Fabaceae) is endemic of the Mediterranean area, known as "Cape broom", but it is also considered as an invasive plant in some places into neotropical area. This plant is also known for its high content of secondary products with multi-pharmacological activity, e.g. antioxidant and phytoestrogenic, which has attracted attention for their potential use in preventive medicine. Thus, the present study was carried out to determine the variation on the DPPH• scavenging capacity and isoflavone content of in vitro seedlings of Cape broom after the stimulus of phytohormones such as indole-3-acetic acid (IAA) and kinetin (KIN), as a biochemical response for future biotechnological studies. Results indicated that the highest germination percentage was achieved in seeds separately exposed to IAA (at 2.5 µM under darkness and 7.5 µM under light) and KIN (at 0.25 µM under light and 0.75 µM under darkness. DPPH• free radical scavenging capacity, total phenolic and total flavonoid contents for in vitro seedlings of G. monspessulana treated with phytohormones were generally higher to that of intact plant. Additionally, in vitro seedlings showed enhanced concentrations of glycosylated isoflavones in the presence of IAA under light exposition. Results indicated that phytohormones (IAA or KIN) affected total phenolic/flavonoid contents, DPPH• scavenging capacity and isoflavone concentrations on in vitro seedlings, which constitutes a biotechnological opportunity to expand the exploitation of isoflavones from G. monspessulana.