The purpose of this study was to examine the characteristics of acetaminophen (APAP)-induced liver damage, using fluorescence bioimaging, serum biochemistry, and histopathology. At six weeks of age, eighteen mice were divided into three groups as group 1 (G1) as control, group 2 (G2) as fluorescence probe control and group 3 (G3) as APAP-treated. G3 mice were orally treated with APAP (800 mg/kg b.w.), while G1 and G2 mice were treated with 0.9% saline. Twenty-two hours after APAP treatment, G2 and G3 mice were intravenously treated with Annexin-Vivo 750 as probe, while G1 mice were treated with saline. Fluorescence bioimaging was performed at two hours after probe treatment. The mice were sacrificed and serum levels of aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase and lactate dehydrogenase were analyzed. Liver damage was examined by hematoxylin and eosin (H&E) staining and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. In vivo bioimaging, fluorescence intensity of the region of interest (ROI) was significantly increased in the livers of G2 and G3 mice compared with those in G1 mice (p<0.05 and p<0.01). In addition, ex vivo bioimaging confirmed that the fluorescence intensity of the ROI was significantly increased in the livers of G2 and G3 mice compared with those in G1 mice (p<0.05 and p<0.01). All examined serum parameters of G3 were significantly increased compared with G1 and G2 (p<0.05 and p<0.01). H&E examination showed acute hepatic cell necrosis in the livers of G3 mice, while there was no cell death in the livers of G1 and G2 mice. TUNEL staining also showed many cell death features in G3 mice, whereas no pathological findings were shown in G1 or G2 mice. In summary, fluorescence bioimaging showed the possibility of cell death detection in the livers of mice treated with APAP, and this was corroborated by serum chemistry and histopathological examination.