A novel HPTLC-densitometry method was developed for separation and quantitative determination of four flavonoids: quercetin 3-O-β-d-glucuropyranoside (miquelianin; QG), quercetin 3-O-β-d-glucopyranoside (isoquercitrin; IQ), quercetin 3-O-β-d-galactopyranoside (hyperoside; HYP) and quercetin 3-O-β-d-(6″-α-l-rhamnosylo)-glucopyranoside (rutin; RUT) in ethyl acetate fractions from aerial parts of selected Potentilla species: P. argentea, P. erecta, Dasiphora fruticosa (syn. P. fruticosa), Drymocallis rupestris (syn. P. rupestris), P. nepalensis var. 'Miss Wilmott' and P. thuringiaca. For the first time, separation of this type of flavonoids was achieved on a HPTLC DIOL F(254) plates using a mixture consisting of ethyl acetate/methyl ethyl ketone/diisopropyl ether/formic acid (3:10:4:1, v/v/v/v). QG, IQ, HYP and RUT were determined by densitometry at 363 nm. Sensitivity, accuracy (recovery rates were between 95.0 and 101.4%) and precision (in both cases intra-day precision and inter-day precision were ≤ 8.0%) of the method were determined. Their amounts were calculated using the regression equations of the calibration curves which were linear in a range of 0.025-0.200 μg/spot for all investigated compounds. The amounts of marker compounds in ethyl acetate extracts of Potentilla species measured by the method ranged between 16.7 ± 1.1 and 41.7 ± 0.6 mg/g for QG, 15.8 ± 1.3 and 36.7 ± 1.0mg/g for IQ, 14.5 ± 0.5mg/g for HYP and 6.7 ± 0.3 and 27.8 ± 2.1mg/g for RUT. The method was found to be relatively simple, specific, precise and accurate and may be used for the quality control of simultaneous determination of quercetin derivatives in Potentilla extracts but also in other similar plant materials.