Amino acid analysis is commonly used to study the nutritional requirements of mammalian cell cultures and to develop improved cell culture media and feeds for the production of biopharmaceuticals.  This analysis is typically performed in an off-line, iterative manner where samples are investigated after the experiment is over.  To provide a real-time assessment of the nutritional requirements and metabolic behavior of cell lines in culture, we have recently integrated an online HPLC system into our standard bioreactor setup.  As well as implementing a simpler assay which does not require amino acid derivatization, this system allows the implementation of feedback control to maintain the desired concentrations of culture nutrients.  Medium and feed development strategies and the resulting cell culture data and process improvements will be discussed.