Summary.The study describes the solvent extraction procedure and improved version of TLC method for the determination of rhaponticin and deoxyrhaponticin in rhizomes and petioles of two species of Rheum L. genus. Extracts from underground parts and petioles of plant were separated on thin layers of silica using a mixture of dichloromethane-95% ethanol-methanol (8:1:1). The bands of rhaponticin and deoxyrhaponticin were satisfactorily separated from the remaining extract components permitting their densitometric quantitative and qualitative determination. The concentration of these compounds in Rheum undulatum was higher than in Rheum rhaponticum. The amount of rhaponticin and deoxyrhaponticin ranged between 0.3 and 31.5 mg/g.