BACKGROUND Chemically defined media (CDM) are used routinely in industrial settings for the production of biopharmaceuticals from mammalian cells, so attention has shifted to understanding their chemical behavior and impact on process robustness. In this context, one particular problem is the risk of cross reactivity and instability in complex mixtures of chemicals at high concentrations. RESULTS In order to characterize potentially unstable medium compounds in feed media, we developed and validated a novel liquid chromatography tandem mass spectrometry (LC‐MS/MS) method to simultaneously quantify the majority of medium compounds. We used this application advantage to provide the following insights into molecular processes occurring in CDM prepared at different temperatures and during storage: Concentration profiles of critical compounds in CDM are independent of preparation temperature and storage material but show significant alterations over time. In particular l‐cysteine, l‐proline, vitamin B6, thiamine and cyanocobalamin were found to be rather unstable. Using mixed mode chromatography allows expansion of the method to cover additional analytes of interest, as demonstrated by inclusion of seven additional CDM reaction products. CONCLUSION The successful development of a new LC‐MS/MS method allowed us to show the impact of storage duration on the stability of CDM compounds. Furthermore, the results suggest that essential parameters for medium preparation (e.g. temperature) and storage upscaling (e.g. vessel material) are not impacting upon the chemical composition of CDM. © 2018 The Authors. Journal of Chemical Technology & Biotechnology published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.