Cell culture media (CCM) used in industrial biotechnology are complex mixtures of molecules and elements that are inherently difficult to analyze comprehensively. CCM quality analysis is of utmost importance for efficient production of protein-based therapeutics with the correct Critical Quality Attributes (CQAs). Here we discuss the use of rapid spectroscopic methods for routine screening of CCM molecular variance which include electronic (UV–vis absorption and fluorescence) and vibrational (Raman, FT-IR, and Near-Infra-red) spectroscopies. CCM analysis needs to provide: identity testing, compositional variance analysis (i.e. lot-to-lot variation), validation of media preparation protocols, and correlations with Critical Process Parameters (CPPs) or product CQAs. Rapid spectroscopic methods can fulfil some of these requirements but only with correct sample handling and preparation. Accurate analysis requires the use of chemometrics combined with a detailed knowledge of sample behavior such as water absorption and chemical stability.