To establish the method for determination of chlorogenic and caffeic acid contents in Prunella vulgaris L., and to investigate the anti-proliferative effect of Prunella vulgaris L. extract on thyroid cancer cells. With chlorogenic and caffeic acids as the quantitative determination targets, their contents in Prunella vulgaris L. are determined by HPLC. Chromatographic column used is Kromasil C18 (250 mm × 4.6 mm, 5 μm), and gradient elution mode is employed. Mobile phase: A is 0.5% phosphoric acid in methanol, and B is acetonitrile, gradient elution: 0~45 min 5%~10% (acetonitrile); flow rate is 1.0 ml/ min; detection wavelength is 323 nm; and column temperature is 20°C. After 48 h treatment of thyroid cancer K1 cells with different concentrations of Prunella vulgaris L. extract, half maximal inhibitory concentration (IC50) of Prunella vulgaris L. extract against K1 cells is calculated by MTT assay. After 48 h treatment of K1 cells with drugs in various groups, morphological changes of K1 cells are observed under inverted phase contrast microscope. Expression of c-myc protein in each group is detected by Western blot. Linear range of chlorogenic acid is 0.32~1.6 μg, r=0.9996, while linear range of caffeic acid is 0.48~2.4 μg, r=0.9998. Sample recovery is 97.6%, RSD=1.5% for chlorogenic acid, and 96.6%, RSD=1.6% for caffeic acid. IC50 of Prunella vulgaris L. extract against K1 cells is 2 mg/ml. Expression of c-myc protein is significantly lower in Prunella vulgaris L. extract groups than in the negative control group (P?0.05). The method proposed herein is accurate, simple and fast, which is suitable for quantitative analysis of Prunella vulgaris L. Prunella vulgaris L. has rather marked inhibitory effect on proliferation of thyroid cancer cells, which provides a theoretical basis for its use in the treatment of thyroid cancer.