Purpose <p>This study aimed to evaluate the capacity of low-field nuclear magnetic resonance (LF NMR) as a standalone or orthogonal method for monitoring key parameters of interest for upstream cell culture fermentation in vaccine manufacturing.</p> Methods <p>Off-line near-infrared (NIR) and NMR measurements of fermentation samples from cells were acquired using the Matrix-F spectrometer with an Excalibur XP25 probe and the Fourier 80 LF NMR spectrometer respectively. Comparative analyses and multivariate modeling were employed against off-line reference and NIR models to characterize the process parameters of glucose, lactate and pH. Additional metabolites were identified and quantified using LF NMR signals. Indirect hard modeling was applied using LF NMR data to achieve relative quantification and visualization of metabolite concentrations.</p> Results <p>LF NMR was applied to quantify lactate, glucose and pH in the cell upstream fermentation media, showing good agreement and comparable measurements to off-line and NIR methods. Further analysis of the LF NMR spectra allowed identification and quantification of additional metabolites. The results demonstrate that LF NMR can provide complementary information to NIR and expand process understanding by revealing dynamic changes in parameters of interest during fermentation.</p> Conclusions <p>This study demonstrates that LF NMR is a feasible and effective analytical tool for monitoring cell fermentation process during vaccine manufacturing. As a standalone or orthogonal method to NIR, LF NMR provides valuable insights to support real-time measurements for process control in vaccine manufacturing.</p> Graphical Abstract <p>Schematic of fermentation sampling using NMR and NIR to predict parameters of interest using predictive modelling. NMR serves as an orthogonal method to predict fermentation parameters as a standalone method and as a reference method for NIR modelling. Created in BioRender. Gerzon, G. (2026) https:// BioRender.com/qgxul4z.&#xa0;</p> <p></p>