The quantitative determination of valepotriates in Valeriana officinalis L. var. sambucifolia Mikan hairy roots by a new high performance liquid chromatographic (HPLC) method is reported. The quantification is based on the simultaneous isocratic determination of the monoene and diene derivatives at one wavelength (208 nm). The analysis is rapid and highly reproducible, giving results similar to those obtained with a currently used HPLC method in which valepotriate quantification is undertaken using two separate runs.