The invention relates to a method for detecting atractyloside and/or hydroxyl atractyloside by virtue of high performance liquid chromatography. The method comprises the following step: by taking an atractyloside and/or hydroxyl atractyloside solution as a reference substance, detecting under a chromatographic condition that octadecyl silane bonded silica gel is taken as an immobile phase and a phosphoric acid-containing brine solution as a moving phase, wherein the moving phase is a phosphate aqueous solution-acetonitrile or a phosphate aqueous solution-methanol in a volume ratio of (75-50): (25-50), the detection wavelength is 200+/-10nm and the phosphate aqueous solution is an aqueous solution containing 0.03-0.5% of tetrabutyl ammonium hydroxide, wherein the pH value is 2.5-5.0. The method provided by the invention is excellent in separating effect, simple and convenient and good in repeatability, and an efficient detection method for quality control of cocklebur fruits comprising atractyloside and/or hydroxyl atractyloside as well as Chinese patent medicines thereof.