Abstract: OBJECTIVE To establish a method for simultaneous determination of phydroxybenzoic acid, vanillic acid, ferulic acid and rutin absorbed into blood of sparganii Rhizoma. METHODS The analysis was carried out by HPLC, chromatographic column:Agilent Eclipse XDB-C₁₈(250 mm×4.6 mm, 5 μm); the mobile phase A was 0.1% phosphoric acid aqueous and solution B was methanol, and the gradient elution HPLC method was used; the flow rate was 1.00 mL·min^-1; the detection wavelength was 254 nm; the column temperature was 30℃. RESULTS The four components showed good separation and good linear relationship in the concentration range of 0.000 5-0.25 mg·mL^-1. The precision of serum sample in each group was good(RSD<10%), the recovery rate was >90%, and the stability of each group was good(RSD<10%), which satisfied the requirements of pharmacokinetics. CONCLUSION The method can determine the four active contents of sparganii Rhizoma in serum simultaneously, accurately and rapidly.