The dynamics of intracellular amino acid pools were determined in batch cultures of Saccharomyces cerevisiae. Immediate termination of metabolic activity was found to be necessary for accurate quantification of in vivo concentrations of intracellular amino acids, due to significant changes in most intracellular amino acid pools observed during extraction without an instantaneous stop of the metabolism. The method applied to batch-cultures of S. cerevisiae on glucose revealed complex dynamics in intracellular amino acid pools. The most drastic changes were observed during the diauxic shift and at the entry into the stationary phase. Even during phases of exponential growth on glucose and ethanol. cells showed significant variations in intracellular amino acid concentrations. The method presented can be used to investigate the physiology of yeast cultures, including industrially relevant batch and fed-batch processes.