In the post-genomic era, increasing efforts have been made to describe the relationship between the genome and the phenotype in cells and organisms. It has become clear that even a complete understanding of the state of the genes, messages, and proteins in a living system does not reveal its phenotype. Therefore, researchers have started to study the metabolome (or the metabolic complement of functional genomics). Within this context, mass spectrometry (MS) has increasingly occupied a central position in the methodologies developed for determination of the metabolic state. This review is mainly focused on the status of MS in the metabolome field, trying to direct the reader to the main approaches for analysis of metabolites, reviewing basic methodologies in sample preparation, and the most recent MS techniques introduced. Apart from the description of the different methods, this review will try to state a general comparison between the several different techniques that involve MS and metabolite analysis, and will highlight their limitations and preferred applicability.