Drug induced toxicities account for nearly half of the cases of acute liver failure in adults over age 50 in the US. Acetaminophen (APAP) is a commonly used non-prescription drug that can be purchased in drug stores and supermarkets. Cases of unintentional APAP overdose and associated hepatotoxicity have been reported to the FDA. Early detection of hepatotoxicity caused by APAP is of great interest to both the scientific and public communities. Therefore, in this study SELDI based analysis of mouse serum following APAP administration was conducted to demonstrate the feasibility of identifying hepatic toxicity biomarkers from a readily available body fluid. Late hepatotoxicity caused by APAP was detected by SELDI. Furthermore, early stage hepatotoxicity caused by APAP that was not detected pathologically was clearly discernable using SELDI. Our results suggest that SELDI analysis can be used for early detection of hepatotoxicity based on the expression pattern of the biomarker peaks.