Identifying and quantifying metabolites in complex biological samples is one of the most challenging aspects of metabolomics. Recently, several important advances in databases, software, instrumentation, and laboratory techniques have greatly simplified the most laborious tasks of metabolite identification and have made quantification more reliable. These technological advances have made bioanalytically oriented studies a feasible alternative to the statistics-based methods commonly used for metabolomics. We discuss the tools that have become most important in our own research and comment on emerging technologies that may play an important role in future studies. In addition, we provide practical guidelines for designing studies and give the step-by-step protocols used in our lab for sample preparation, metabolite identification, and accurate quantification of molecules.