Abstract: This study aimed to establish an efficient 2-DE proteomic method of H.parasuis.The 2-DE reaction suitable for the determination was established and optimized through the formulation of lysis buffer,modification on sample preparation,adjustment on sampling,and time needed for isoelectric focusing determination.Two clinical isolates of H.parasuis were used inthe comparative analysis.The results showed that high-resolution,highrepeatability 2-DE pages could be obtained with 500μg protein samples extracted by the ultrasound-lysis(7mol·L-1 ultra lysis buffer)-centrifugation method and purified by a 2-D clean up kit.They were separated by isoelectric focusing on the first dimension reaching 60 000 Vh,and visualized by silver-staining.