Abstract Cell culture media are very complex chemical mixtures that are one of the most important aspects in biopharmaceutical manufacturing. The complex composition of many media leads to materials that are inherently unstable and of particular concern, is media photo-damage which can adversely affect cell culture performance. This can be significant particularly with small scale transparent bioreactors and media containers are used for process development or research. Chromatographic and/or mass spectrometry based analyses are often time-consuming and expensive for routine high-throughput media analysis particularly during scale up or development processes. Fluorescence excitation–emission matrix (EEM) spectroscopy combined with multi-way chemometrics is a robust methodology applicable for the analysis of raw materials, media, and bioprocess broths. Here we demonstrate how EEM spectroscopy was used for the rapid, quantitative analysis of media degradation caused by ambient visible light exposure. The primary degradation pathways involve riboflavin (leading to the formation of lumichrome, LmC) which also causes photo-sensitised degradation of tryptophan, which was validated using high pressure liquid chromatography (HPLC) measurements. The use of PARallel FACtor analysis (PARAFAC), multivariate curve resolution (MCR), and N-way partial least squares (NPLS) enabled the rapid and easy monitoring of the compositional changes in tryptophan (Trp), tyrosine (Tyr), and riboflavin (Rf) concentration caused by ambient light exposure. Excellent agreement between HPLC and EEM methods was found for the change in Trp, Rf, and LmC concentrations.