Our work focuses on the quality control and structural identification of Photocyanine as a cancer therapeutic photosensitizer.  Photocyanine is a mixture which contains four ZnPcS2P2 type substituted Phthalocyanine isomers.  In order to obtain the single component from Photocyanine, the mixture of four isomers possessing the similar structures and chemical property had been isolated and purified.  An HPLC method with a mixture of methanol-acetonitrile-ion-pair buffer as the mobile phase was applied to isolate the four isomers by means of a semi-preparative C₁₈ column.  To remove the salts which were mixed in the preparative product, a SPE C₁₈ column was used to separate the salts by elution with water and then the marker component was eluted by methanol.  Subsequently, a column of Sephadex LH-20 gel was applied to elute the crudes with methanol to desalination.  The purity of the isolated compound was measured by TLC and four different isomers of phthalocyanine were obtained.  The chemical structures of them were elucidated by ¹H NMR spectra, gCOSY and NOE1D.  An HPLC-DAD method was developed for simultaneously determination of four major isomers in Photocyanine with a C₁₈ column (Grace Smart, 150 mm × 4.6 mm ID, 5 μm).  The separation was carried out with a gradient program at a flow rate of 1.0 mL·min⁻¹.  The mobile phase was a mixture of acetonitrile and ion-pair buffer (0.01 mol·L⁻¹ hexadecyl trimethyl ammonium bromide and 0.01 mol·L⁻¹ potassium dihydrogen phosphate, adjusted the pH value to 6.8 with potassium hydroxide solution).  The resolution values of four isomers were 2.5, 1.20, 1.33, and 1.8.  Linear regression analysis for four compounds was performed by the external standard method.  Four constituents were linear in the concentration range of 0.005 to 10 μg.  The values of relative standard deviation (RSD) of intra-day were 0.12%, 0.66%, 0.99%, and 1.21%, respectively.  The limits of detection for four compounds were 15 ng, 20 ng, 12 ng, and 25 ng, respectively.  This method was simple, accurate and reproducible.  The developed method can be successfully applied to analyze isomers in Photocyanine.