Changing the protein source of fish feed from fish meal to alternative sources of protein will affect traits such as fish growth, quality, and feed utilization. The present investigation was initiated to introduce a two-dimensional gel electrophoresis based proteomic workflow as a tool to investigate feed effects on fish by analyzing protein changes in the fish gut. The workflow was used to study the effect of substituting fish meal in fish feed by alternative sources of protein. Rainbow trout divided into five groups were fed for 72 days with feeds varying in protein composition. By two-dimensional gel electrophoresis proteins extracted from the pyloric ceca were separated, making it possible to measure the abundance of more than 440 protein spots. The expression of 41 protein spots was found to change due to differences in feed composition. By mass spectrometry 31 of these proteins were identified, including proteins involved in digestion (trypsinogen, carboxylic ester hydrolase, and aminopeptidase). The many expression changes indicated that the trout, when adapting to differences in feed formulation, alter the protein composition of the gut.