Proper liver function is crucial for metabolism control and to clear toxic substances from the bloodstream. Many small-molecule therapeutics accumulate in the liver, negatively impacting liver function and often resulting in hepatotoxicity and cell death. Several analytical methods are currently utilized to evaluate hepatotoxicity and monitor liver function. To date, none of these methods have specifically targeted protein phosphorylation-mediated signal transduction pathways which should be altered in response to toxic effects of small molecule therapeutics. To develop novel assays to probe specific signaling pathways in the liver, identification and quantification of specific protein phosphorylation sites in this complex organ is necessary. Here, we have utilized an optimized immobilized metal affinity chromatography (IMAC) protocol to enrich phosphorylated peptides from a tryptic digest of proteins isolated from whole liver lysate. LC-MS/MS analysis of IMAC-enriched peptides resulted in the identification of more than 300 phosphorylation sites from over 200 proteins in rat liver, a significant advance over previously published analyses of the liver phosphoproteome. Previously characterized phosphorylation sites and potentially novel sites were identified in the current study, including sites on proteins implicated in metabolism regulation, transcription, translation, and canonical signaling pathways. Moreover, protein phosphorylation analysis was performed without prior fractionation of the sample, enabling analysis of small sample amounts while minimizing analysis time, potentially allowing for high-throughput assays to be performed with this methodology. From these data, it appears that this methodology can be used to identify new phosphorylation sites and, in combination with a stable isotope-labeling step, to investigate the effects of liver diseases, cancer and evaluate potential toxicology of new drug substances.