Development of a method to elucidate changes in bovine pancreatic cell function using proteomic tools is described. Two groups of 48 and 45 crossbred steers in years 1 and 2 respectively were fed a high-moisture corn-based diet during finishing and were ranked according to their pancreatic [alpha]-amylase activity. Steers with high, medium or low [alpha]-amylase activity were selected, with 3 for each activity range and a total of 9 for each experimental year, for comparison of their proteome profiles. Pancreatic samples from each animal were fractionated using two-dimensional high-performance liquid chromatography and detected using UV spectrophotometry. Software analysis revealed 119 common protein fractions among the 18 animals, and statistical analysis revealed 10 of these fractions differing in abundance between animals with high and low pancreatic [alpha]-amylase activity. Six protein fractions identified after tandem mass spectrometry analysis and database searches were found to have either protein-binding, DNA-binding or enzymatic capabilities.