Currently, liquid chromatography-mass spectrometry (LC-MS) is one of the most important analytical technologies for detecting hundreds of metabolites in the field of metabolomics. A recent advance in LC that has impacted metabolomics is the development of UPLC (ultra-performance liquid chromatography). In this chapter, we describe the analytical methodologies for the global metabolic profiling of serum, urine, and tissue samples using UPLC-Q-TOF (quadrupole-time-of-flight)-MS. Aqueous metabolites are extracted after adding methanol/acetonitrile/acetone and then analyzed by UPLC-MS under positive and/or negative ionization mode. With the aid of multivariate statistical analysis, separation between various groups can be observed in the score plots, and biomarkers are screened in the loading/ weight/VIP (variable importance in the projection) scatterplots. Furthermore, putative markers can be identified through comparison with the authentic standards based on tandem mass spectrometry (MS/MS) fragmentation pattern and LC retention. We expect that our protocol, with modifications if necessary, can be useful in many metabolomics studies and a wide range of research areas related to small molecules and LC-MS.