OBJECTIVE：To establish a method for the content determination of tetra-substituted tetraphenylporphyrin derivative （TLTAPP）. METHODS：RP-HPLC was performed on the column of Waters SunFireTM C18 with the mobile phase A of acetonitrile-water （containing 0.1％ trifluoroacetic acid，95 ∶ 5，V/V） and mobile phase B of acetonitrile-water（containing 0.1％ trifluoroacetic acid，5 ∶ 95，V/V） （gradient elution） at a flow rate of 1.0 ml/min； the column temperature was 30 ℃， the detection wavelength was 418 nm and the injection volume was 20 μl. RESULTS： The linear range of TLTAPP was 1.55-95 μg/ml（r＝0.999 4）； the detection limit and quantitation limit were 1.1 ng and 3.8 ng； RSDs of precision， reproducibility and stability tests were lower than 1％；recovery was 99.00％-101.60％（RSD＝0.89％，n＝9）. CONCLUSIONS： The method is simple， rapid， accurate and specific， and can be used for the content determination of TLTAPP.