The sulfur metabolic pathway plays a central role in cell metabolism. It provides the sulfur amino acids methionine and cysteine, which are essential for protein synthesis, homocysteine, which lies at a critical juncture of this pathway, S-adenosylmethionine, the universal methyl donor in the cell, and glutathione (GSH), which has many crucial functions including protection against oxidative stress and xenobiotics. The intracellular level of these metabolites, which are closely connected with other cellular metabolic pathways, is of major importance for cell physiology and health. Three mass spectrometry-based methods for the determination of sulfur metabolites and also related compounds linked to the glutathione biosynthesis pathway are presented and discussed. The first one enables absolute quantification of these metabolites in cell extracts. It is based on liquid chromatography-electrospray triple quadrupole mass spectrometry coupled to (15)N uniform metabolic labeling of the yeast Saccharomyces cerevisiae. The two other methods are global approaches to metabolite detection involving a high-resolution mass spectrometer, the LTQ-Orbitrap. Ions related to metabolites of interest are picked up from complex and information-rich metabolic fingerprints. By these means, it is possible to detect analytical information outside the initial scope of investigation.