OBJECTIVE The aim of the study is to measure partition coefficients of two highly hydrophobic compounds， icariside-Ⅱ and icaritin， based on their retention time on HPLC. METHODS A series of compounds with similar structure were selected as the reference substances and their partition coefficients were determined by shake-flask method or obtained from the science finder database. The retention time of the above reference substances was determined under different concentrations of organic phase. Logk_w was obtained by extrapolating to zero concentration of organic phase. A linear relationship between lgP and lgk_w was established. Logk_w of two substances for validation purpose (quercetin and formononetin) and two substances to be measured (icariside-Ⅱ and icaritin) were obtained by the same method. LogP of above four compounds was calculated from the linear regression equation obtained from the reference substances and compared with the lgP of validation substances determined by shake-flask method. RESULTS A linear regression equation between lgP and lgk_w was established： lgP=(1.532±0.091)lgk_w-(1.805±0.301)， r²=0.959 0. The lgP of validation substances determined by shake-flask method is within the range of lgP measured by HPLC method， indicating that the partition of the selected compounds in HPLC system is similar to that in octanol-water system. Therefore， the partition coefficient could be determined using their retention time on HPLC. The calculated lgP value of icariside-Ⅱ and icaritin is （5.114±0.712） and （6.074±0.769）， respectively. CONCLUSION HPLC is an accurate， convenient and rapid method to determine the partition coefficient of highly hydrophobic compounds (lgP>4).