Technical Abstract: An HPLC method permitting the simultaneous determination of fourteen compounds (phenylalkanoids and monoterpenoids) from the roots of Rhodiola rosea was developed. A separation was achieved within 35 minutes by using C-18 column material, a water/acetonitrile mobile phase, both containing 0.05% phosphoric acid gradient system and a temperature of 53°C. The method was validated for linearity, repeatability, limits of detection (LOD) and limits of quantification (LOQ). The limits of detection and limits of quantification of fourteen phenylalkanoids and monoterpenoids were found to be in the range from 0.20-1.0 µg/mL and 0.5-3.5 µg/mL, respectively. The wavelength used for quantification of phenylalkanoids and monoterpenoids with a diode array detector were 205, 220 and 251 nm. The method was used to analyze the roots of two species of Rhodiola and commercial extracts of R. rosea and provides preliminary evidence of phytochemical differences between North American and Eurasian populations of R. rosea. LC-mass spectrometry coupled with electrospray ionization (ESI) interface method is described for the identification of phenylalkanoids and monoterpenoids in various Rhodiola samples. This method involved the use of the [M+H]+, [M+NH4]+, and [M+Na]+ ions in the positive ion mode with extractive ion monitoring (EIM).