Introduction: Leguminous plants open their leaves during the day and close them at night as if sleeping, a type of movement that follows circadian rhythms, and is known as nyctinastic movement. This phenomenon is controlled by two endogenous bioactive substances that exhibit opposing activities: Leaf-Opening Factor (LOF), which opens the leaves, and Leaf-Closing Factor (LCF), which closes them. Potassium isolespedezate (1) is LOF for Cassia obtusifolia and other species of the genus Cassia. We report on the detection and identification of the target proteins of 1 using the stepwise FLAG-tagging strategy. Materials and Methods: First, we labeled the target protein with azide group by incubating the iodoacetamide-type probe (2), which is the target cell of 1, and then, succeeded in introduction of FLAG using click chemistry. Results and Discussion: By using this method, the 83 kDa cytoplasmic protein (CTPL; cytosolic target protein of lespedezate 1) was detected and purified as a target protein of 1. This protein was identified as MetE (5-methyltetrahydropteroyltriglutamate ?homocysteinemethyltransferase). Conclusion and Prospects: We developed stepwise FLAG-tagging strategy for purification of target protein of bioactive substances. Our results suggested that our strategies have a possibility to d