A high-performance liquid chromatographic (HPLC) technique coupled with photodiode array (PDA) detection has been proposed for simultaneous determination of five flavonoids, i.e. quercetin 3-O-β-d-glucopyranoside, quercetin 4′-methoxy-3-O-β-d-galactopyranoside, kaempferol 3-O-β-l-rhamnopyranoside, asebotin, and kaempferol 7-methxoy-3-O-α-l-rhamnopyranoside in extract of the whole plant of Saussurea mongolica Franch. The optimum conditions for separation were achieved on a 4.6 × 250 mm i.d., 5-μm particle, C₁₈ column with acetonitrile and 1% acetic acid (20:80, v/v) as the mobile phase at a flow rate of 1.0 mL min⁻¹. For all the analytes, a good linear regression relationship (r of >0.999) was obtained between peak area and concentration over a relatively wide range. The method was validated for repeatability, precision, stability, and accuracy. Seven different extraction procedures were investigated for preparation of the sample solution. The validated method was successfully applied to simultaneous analysis of these flavonoids in S. mongolica and was found to be simple and efficient.