This study compares the total liver proteome of inbred alcohol-preferring line (iP) rats exposed to alcohol with iP rats without alcohol experience. Rat liver proteins were extracted using a three-step procedure. Each of the three solutions solubilizes a different set of proteins. The extracted proteins were separated by 2-DE. Scanned gels of two sample groups, alcohol-exposed iP and alcohol-naïve iP, were compared, revealing many protein spots with significantly higher or lower densities. These spots were cut from the gel, destained, and subjected to trypsin digestion and subsequent identification by LC-MS/MS. Twenty-four individual rats, 12 alcohol-naïve, and 12 alcohol-exposed, were used in this study. Two groups, each containing six naïve and six exposed animals, were created for statistical comparison. For the first group, 64 spots were observed to have statistically significant intensity differences upon alcohol exposure across all three extracts while 118 such spots were found in the second group. There were 113 unique proteins in both groups together. The majority of these proteins were enzymes. Significant changes are observed for three major metabolic pathways: glycolysis, gluconeogenesis, and fatty acid beta-oxidation. In addition, enzymes involved in protein synthesis and antioxidant activity show significant changes in abundance in response to alcohol exposure.