Chemically defined media have been widely used in the biopharmaceutical industry to enhance cell culture productivities and ensure process robustness. These media, which are quite complex, often contain a mixture of many components such as vitamins, amino acids, metals and other chemicals. Some of these components are known to be sensitive to various stress factors including photodegradation. Previous work has shown that small changes in impurity concentrations induced by these potential stresses can have a large impact on the cell culture process including growth and product quality attributes. Furthermore, it has been shown to be difficult to detect these modifications analytically due to the complexity of the cell culture media and the trace level of the degradant products. Here, we describe work performed to identify the specific chemical(s) in photodegraded medium that affect cell culture performance. First, we developed a model system capable of detecting changes in cell culture performance. Second, we used these data and applied an LC-MS analytical technique to characterize the cell culture media and identify degradant products which affect cell culture performance. Riboflavin limitation and N-formylkynurenine (NFK), a tryptophan oxidation catabolite, were identified as chemicals which results in a reduction in cell culture performance. (c) 2015 American Institute of Chemical Engineers Biotechnol. Prog., 32:74-82, 2016