The identification of pancreatic proteins is generally hampered by the high content and activity of proteases produced by this organ. The aim of this work was the development of a protocol for the analysis of pancreatic juice by two-dimensional (2-D) gel electrophoresis allowing consistent and reproducible protein analysis encompassed by high-resolution protein 2-D maps and subtle protein spot recognition without substantial losses due to proteases. Immobilized pH gradient (IPG) strips were used for the first dimension, the second dimension was performed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). However, the key step was the sample preparation technique. Improvements were achieved by using several protease inhibitors (phenylmethylsulfonyl fluoride, aprotinin, L-1-chloro-3-[4-tosyl-amido]-7-amino-2-heptanine (TLCK)-HCI, Complete) to prevent degradation of the proteins. The application of different pH-ranges was a valuable step for getting an overview of the expressed protein pattern. These investigations resulted in well-resolved 2-D maps with a high reproducibility.