The rapid and inexpensive analysis of the complex cell culture media used in industrial mammalian cell culture is required for quality and variance monitoring. 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. We have shown that the methodology can identify compositional changes and predict the efficacy of media in terms of downstream titer . Here we describe how to extend the measurement methodology for the quantification of tryptophan (Trp), tyrosine (Tyr) in complex chemically defined media. The sample type is an enriched basal RDF medium in which five significant fluorophores were identified: Trp, Tyr, pyridoxine, folic acid, and riboflavin. The relatively high chromophore concentrations and compositional complexity lead to very significant matrix effects which were assessed using PARAllel FACtor analysis2 (PARAFAC2). Taking these effects into account. N-way partial least squares (NPLS) combined with a modified standard addition method was used to build calibration models capable of quantifying Trp and Tyr with errors of similar to 4.5 and 5.5% respectively. This demonstrates the feasibility of using the EEM method for the rapid, quantitative analysis of Trp and Tyr in complex cell culture media with minimal sample handling as an alternative to chromatographic based methods. (C) 2012 Elsevier BM. All rights reserved.