The pharmacological activities of the flavonoids show the interest in quantifying these constituents in phytopharmaceutical preparations, as well as in the validation of the analytical methodologies. LC methods have been reported to quantify isolated flavonoids or these compounds in complex biological matrices, such as herbal raw materials and extractive preparations. This work was designed, therefore, to develop an LC system to separate quercetin, luteolin and 3-O-methylquercetin and to quantify them in extractive solutions from Achyrocline satureioides. The main validation parameters of the method are also determined. The method showed linearity for quercetin and luteolin in the range 1-10 microg/ml. The aqueous and ethanol 80% extractive solutions showed linear response in the range 2.5-20 microl/ml and ethanol 40% extractive solution in the range 2.5-10 microl/ml. Precision and accuracy were determined for ethanol 80% extractive solution, in the concentration of 10 microl/ml. The LC method showed an excellent performance in separating the flavonoids quercetin, luteolin and 3-O-methylquercetin in A. satureioides extracts, since the presence of interference has been previously evaluated.