OBJECTIVE To establish a method for determining the entrapment efficiency of alprostadil injection, compare the differences between ultrafiltration centrifugation and microcolumn centrifugation methods for determining the entrapment efficiency of alprostadil injection, and select the optimal method. METHODS High pressure homogenization method was used to prepare alprostadil injection. Ultrafiltration membrane with different molecular weights were used to establish the ultrafiltration centrifugation method by selecting the centrifugation speed and time. The micro column was prepared with Sephadex G-50, and the encapsulated and free drugs were separated by centrifugal elution. The separation effect was evaluated by establishing the elution curve of soybean oil and free alprostadil. At the same time, the difference between different elution solvents was investigated, the optimal elution solvent was screened, and the microcolumn centrifugation method was established. The drug content was determined by high-performance liquid chromatography and post column derivatization device, and the entrapment efficiency was calculated. RESULTS The entrapment efficiency of three batches of alprostadil injection measured by ultrafiltration centrifugation method ranged from 98.6% to 99.5%, while the entrapment efficiency measured by microcolumn centrifugation method ranged from 89.5% to 91.3%. CONCLUSION The results of the ultrafiltration centrifugation method for determining the entrapment efficiency are too high and not suitable for the determination of the entrapment efficiency of this product. The microcolumn centrifugation method using glycerol aqueous solution as the eluting solvent is suitable for the determination of the entrapment efficiency of this product.