The direct separation detection of amino acids by anion exchange chromatography with integrated pulsed amperometric detection was optimized for the analysis of typical mammalian cell culture broth samples. Existing gradient elution conditions were adapted, considering the additions of peptone (2 g/L) and 10 vol% fetal calf serum to the medium as well as changing concentrations of glucose from 5.5 g/L up to complete consumption. Samples had to be analyzed in two dilutions with water (1:33.3 and 1:200) due to the strongly varying amino acid concentrations in the samples as a result of the medium composition and cell metabolism. The method was validated in a linear working range for the most common amino acids (2.5-7.5 and 1.25-3.75 microM for cystine/cysteine with 15 microl injection volume). The relative standard deviation of the method for all amino acids was less than 5%, with detection limits of less than 0.6 microM and quantitation limits of less than 1.6 microM. As an example, data for the amino acid composition of different media used for the production of inactivated influenza vaccines in cell culture are shown.