Inorganic polyphosphate (iPoP)-linear chains of up to hundreds of phosphate residues-is ubiquitous in nature and appears to be involved in many different cellular processes. In Saccharomyces cerevisiae, iPoP has been detected in high concentrations, especially after transfer of phosphate-deprived cells to a high-phosphate medium. Here, the dynamics of iPoP synthesis in yeast as a function of the growth phase as well as glucose and phosphate availability have been investigated. To address this question, a simple, fast and novel method for the quantification of iPoP from yeast was developed. Both the iPoP content during growth and the iPoP "overplus" were highest towards the end of the exponential phase, when glucose became limiting. Accumulation of iPoP during growth required excess of free phosphate, while the iPoP "overplus" was only observed after the shift from low- to high-phosphate medium. The newly developed iPoP quantification method and the knowledge about the dynamics of iPoP content during growth made it possible to define specific growth conditions for the analysis of iPoP levels. These experimental procedures will be essential for the large-scale analysis of various mutant strains or the comparison of different growth conditions.