Non-alcoholic fatty liver disease is the most common cause of chronic liver diseases [1]. Liver lipid content has been suggested to play an important pathogenic role in the development of liver fibrosis and cirrhosis [2], but Liver biopsy is still the gold standard for diagnosing and assessing the disease [3]. However, the invasive limited tissue sampling of the biopsy presents problems. Proton magnetic resonance spectroscopy (1H-MRS) allows the study of cellular biochemistry and metabolism, and provides a non-invasive mean to determine disease abnormalities and progression in vivo and longitudinally. 1H-MRS permits longitudinal assessment of fat fraction, saturated and unsaturated [4]. The aim of this study was to characterize hepatic lipid metabolites changes in high-fat diet induced liver steatosis model using in vivo 1H-MRS.This study included 17 male C57BL/6 mouses, 8 high-fat diet (45%) mice for 20 weeks (mean±SD, 40.2±4.58g) and 9 normal mice (28.9±2.22g). A single-voxel 1H-MRS was performed using a PRESS sequence at 9.4T. (e.g., Broker System with a 4-channel rat heart receive-only array animal coil). The examination (voxel size, 2×2×2 mm³) was measured from liver parenchyma in mouse livers with and without high-fat diet, respectively. The typical water peak linewidth (FWHM) ranged from 37 to 69 Hz (mean±SD, 52.1±9.6 Hz). After shimming procedure water suppression was accomplished with "VAPOR" pulses. The spectral acquisition parameters were TR/TE = 2500/16 ms, and 256 acquistions for averaging. A fully relaxed, unsuppressed spectrum was also acquired to measure the water peak (32 averages). LCModel fitting was conducted using experimental basis sets (e.g., lipid-8). Signal integrals of lipid methyl protons (-CH3; 0.9 ppm), methylene protons ((-CH2-)n; 1.3 ppm), allylic protons (-CH2-C=C-CH2-; 2.0 ppm), diallylic protons (=C-CH2-C=; 2.8 ppm), glycerol backbone (CH2-COO); 4.1 ppm), glycerol backbone (CH2-COO; 4.3 ppm), methene protons (-CH=CH-; 5.3 ppm) and protons from choline-containing compounds (CCC; 3.2 ppm) were measured by integrating areas under peaks. Total lipid was quantified by dividing peak of H₂O. In addition, total saturated fatty acid (TSFA), total unsaturated fatty acid (TUFA), total unsaturated bond (TUB), polyunsaturated bond (PB), and unsaturated index (UI) were estimated (Figure 1). After the MR examinations, normal and high-fat diet mice were sacrificed for histological evaluation. Liver specimens were fixed in formalin, embedded in paraffin, sectioned and examined by light microscopy after standard hematoxylin-eosin staining.Figure 2 shows the typical 1H-MRS spectra from a mouse without (a) and with high-fat diet for 20 weeks (b). Figure 3 shows that significant increase in lipid signals at 0.9, 1.3, 2.1, 2.3, 2.8, 4.1, 4.3, and 5.3 ppm was found in mice with high-fat diet (p<0.0001). TL, TUB, UI, and Cho were also increased with high-fat diet (p<0.001, p=0.039, p=0.045, p=0.008). However, no significant differences were observed in TSFA, TUFA, and PB between normal and high-fat diet (p=0.562, p=0.522, p=0.319). Figure 4 shows the typical hematoxylin-eosin staining of normal liver and livers subjected to 20 weeks after high-fat diet. Characteristic histological features of intracellular fat vacuoles were consistently observed in livers with high fat diet. The averaged pathological score level (e.g., steatosis) was found to be significantly higher in high fat diet liver compared to normal liver (p<0.05 for reading 1^st; p<0.001 for reading 2^nd).The present study demonstrated that in vivo 1H-MRS can be used to detect the hepatic lipid metabolite abnormalities in fatty liver disease [5]. The main observation in this work was the significant increase of lipid signals in the liver parenchyma of fatty liver mice with high-fat diet. In this study, the total saturated fatty acid was high in the fatty liver mice, but not significant (p>0.05). The increase in total lipid increase contributed mainly to the total saturated fatty acid increase in mice with high-fat diet, which has been suggested to be related with activated apoptosis induced by saturated fatty acids because lipid-induced cell toxicity [6]. Therefore, 1H-MRS is useful in detecting and characterizing various hepatic lipid alterations at early phase in mouse liver steatosis prior to development of fibrosis.