Abstract Plants secondary metabolites undergoes qualitative and quantitative variation due to environmental and growth factors. It is a crucial factor to select the proper time for collection of medicinal plants to assure maximum content of active components reflected as maximum efficacy. Olive leaves (Olea europaea L.) are known traditionally for their antidiabetic effect. The secoiridoid glycoside oleuropein is the main active component of Olive leaves responsible for the biological activity. The current study was conducted to monitor the seasonal variation of oleuropein in Olives leaves collected from the same location. To achieve this goal a validated HPLC method following the ICH guidelines was established. Separation was conducted using RP18 column and a mobile phase consisted of ultrapure water containing 20% acetonitrile and 1% acetic acid. Detection was performed at 254 nm with 1 mL/min flow rate. The method was simple, linear, accurate, precise, specific and robust. The analyses revealed considerable variations in the level of oleuropein throughout the year. This variation cannot be explained by temperature variation during the year. Two points of high levels of oleuropein were detected prior to flowering stage and ripening of the fruits. The levels of growth regulators most likely is responsible for the increased production of oleuropein. It is recommended that leaves intended for medicinal use to be collected during the fruiting stage prior to fruit ripening.