Three homoisoflavonoids, 6-aldehydo-3-ophiopogonanone A, methyl ophiopogonanone A and ophiopogonanone A from Ophiopogon japonicus were analyzed simultaneously by HPLC with acetonitrile-water containing 0.5% H3 PO4 (58:42) as the mobile phase, and the detection wavelength was set at 296 nm (0-14 min) and 275 nm (14-22 min). The mean recoveries of three homoisoflavonoids were 99.41%-99.86% (RSD 0.82%-1.05%). The linear response ranges of. 6-aldehydo-3-ophiopogonanone A, methyl ophiopogonanone A and ophiopogonanone A were 0.165-0.990 microg (r = 0.999 9), 0.153-0.918 microg (r = 0.999 9), and 0.270-1.620 microg (r = 0.999 9), respectively. This method was certified to be accurate and reliable and can be used for quality control of O. japonicus.