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A simple, rapid and specific method based on cloud‐point extraction (CPE) was developed to determine ampelopsin in rat plasma after oral administration by reversed‐phase high‐performance liquid chromatography. The non‐ionic surfactant Genapol X‐080 was chosen as the extract solvent. Some important parameters affecting the CPE efficiency, such as the nature and concentration of surfactant, extraction temperature and time, centrifuge time and salt effect, were investigated and optimized. Separation was accomplished using a C18column by gradient elution with a acetonitrile–phosphate buffer solution as the mobile phase. The detection wavelength was set at 290 nm. Under optimum conditions, the linear range of ampelopsin in rat plasma was 20–2000 ng/mL (r2=0.9996). The limit of detection was 6 ng/mL (S/N=3) with the limit of quantification being 20 ng/mL (S/N=10). The proposed method has been successfully applied for pharmacokinetic studies of ampelopsin from rat plasma after oral administration.