Corynebacterium glutamicum is a promising candidate for industrial production of methionine as feed additive thereby replacing current chemical processes [1]. To obtain a basis for rational design of C. glutamicum strains for methionine production, an efficient method for the analysis of intracellular metabolites, i.e., intermediates of the product pathway and amino acids such as cysteine as S-donor or serine and glycine involved in the supply of the terminal methyl group, is crucial (Fig. 1). Current methods applied in this context are either laborious or limited to only a few compounds [2], [3] and [4]. The present work provides a sensitive and straightforward HPLC procedure for simultaneous quantification of 19 proteinogenic amino acids and all intermediates of methionine biosynthesis of C. glutamicum. The method is applicable to both culture broth and cell extracts.