Comprehensive two-dimensional gas chromatography-time-of-flight mass spectrometry (GC × GC-TOF MS) is a powerful analytical platform for analysis of metabolites in mouse liver. Metabolites are extracted using a solvent mixture of methanol/water (4:1, v/v). The metabolite extracts are analyzed on GC × GC-TOF MS after derivatization. The experimental data are further processed for metabolite identification and quantification. A three-tier method is used for high-accuracy metabolite identification, while metabolite quantification is performed to recognize metabolites with significant abundance differences between sample groups.