The pentose phosphate pathway (PPP) is a central pathway of the cellular carbohydrate metabolism. The PPP is directly interconnected with glycolysis and composed of a irreversible, oxidative- and a reversible, non-oxidative part. The PPP provides the cell with intermediates for several bioorganic syntheses, and acts as balancer for the redox state in response to oxidant exposures (1,2). Here, we provide a liquid chromatography tandem mass spectrometry (LC-MS/MS) based protocol for accurate quantification of PPP intermediates in S. cerevisiae .