To study the content and clinical effect of Harmine and Harmaline in Hayi wind-dispelling ointment by HPLC method. Methods: The sample was separated on Agilent TC-C18(4.6 mm ×250 mm, 5 μm). CH30H of 0.01 moL·L-1 and(NH3)2SO4(63∶37 pH regulated to 4.0 by triethylamine)were used as the mobile phase, with flow rate at 1 mL·min-1, column temperature at 25 ℃, the determining wavelength at 320nm. Complete random method was used to divide control group and experimental group to compare the efficacy of Hayi wind-dispelling ointment. Results: Harmine calibration curve was linear over the range of 12-83 μg(r=0.9998，n=6). The average recovery rate was 100.14%, RSD was 1.33%. Harmaline calibration curve was linear over the range of 12-83 μg(r=0.9998, n=6). The recovery rate was 98.89%， RSD was 1.77%. The total effective rate of treatment group was 97.3%, followed by the control group at 90.1%.There was significant difference between the two groups(P<0.01), which indicates that the overall curative effect of treatment group was obviously better than that of the control group.Conclusion: The HPLC method is simple, accurate, and reliable, which can be applied to determine the content of harmine and harmaline in Hayi wind-dispelling ointment. Hayi wind-dispelling ointment is effective and safe for the treatment of shoulder, back and leg pain and can reduce the relapse rate of patients, thus can be further promoted in clinical practice.