Abstract: A high- performance liquid chromatography- diode array detection ( HPLC- DAD) method was established for detection the simultaneous determination of 12 kinds anti- fatigue agents, which were illegally added in health care products.The sample were separated on Agilent ZOBAX SB- C18column ( 4.6 mm × 250 mm, 5 μm) , gradient eluted with mixture of acetonitrile and phosphoric acid- triethylamine ( 7 m L triethylamine, dilute with water to1000 m L, with phosphoric acid to regulate p H to 2.8 ± 0.05) at a flow rate of 1.0 m L / min. The column temperature was 18 ℃ and the detection wavelength was set at 230 nm.Results showed that good linearity was obtained for 12 kinds anti- fatigue agents in 12 components in a wide range and correlation was greater than 0.999.The LOD was from 4.02 to 9.17 ng.The recovery rate was from 98.69% to 109.07% and RSD was from 0.20% to 2.46%.Using this method, 1 batch of illegally added chemicals were detected from 38 batches of samples.This method was simple and precise, it can provide a reliable way for detecting 12 Anti- fatigue agents in health care products.