Soluble inositol polyphosphates are implicated in the regulation of many important cellular functions. This protocol to extract and separate inositol polyphosphates from Saccharomyces cerevisiae is divided into three steps: labeling of yeast, extraction of soluble inositol polyphosphates and chromatographic separation. Yeast cells are incubated with tritiated inositol, which is taken up and metabolized into different phosphorylated forms. Soluble inositol polyphosphates are then acid-extracted and fractionated by high-performance liquid chromatography. The radioactivity of each fraction is determined by scintillation counting. This highly sensitive and reproducible method allows the accurate detection of subtle changes in the inositol polyphosphate profile and takes less than 48 h. It can easily be applied to other systems and we have included two adaptations of the protocol, one optimized for mammalian cells and the other for Arabidopsis thaliana.