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Among mammalian cells, the BHK-21 cell line has found applications in veterinarian viral vaccines and heterologous protein production, even in the transient mode. Many media formulations for mammalian cell culture often contain an optional pH indicator, phenol red. This dye provides a useful, visual but qualitative mode for monitoring metabolism within a culture device. The aim of this work was to calibrate a Partial Least Squares (PLS) model for glutamine, glutamate, glucose, lactate, and viable cell concentration based on off-line ultraviolet-visible spectra for BHK-21 suspension cell culture in bioreactors.