Recent advances in the performance of hyphenated technologies based on ultrapressure chromatography and high-sensitivity mass spectrometry have set the stage for a myriad of metabolomics studies in plants and other organisms. In this chapter, we describe the use of a UPLC (Ultraperformance Liquid Chromatography)-qTOF (quadrupole time-of-flight) system for profiling semipolar metabolites in the model fruit plant tomato. An optimized extraction method, instrument parameters and data treatment procedures are provided. The value of UPLC instruments, which use small particle size chromatographic columns, in terms of resolution, separation, and short injection times are presented. When coupled to a TOF mass spectrometer with high resolution and mass accuracy, good dynamic range, and a fast spectral acquisition capacity, this system is most suitable for the extensive profiling of hundreds of plant metabolites.