The development of in-situ monitoring techniques enabling the real-time acquisition of information concerning the key variables over different Escherichia coli cultivation conditions and strategies is a crucial step towards the optimization of a plasmid bioproduction process. This work shows the use of a Near-InfraRed (NIR) fiber optic probe immersed in the culture broth for the real-time acquisition of NIR spectra along different E. coli cultures conducted with mixtures of the carbon sources glucose and glycerol, and performed in both batch and fed-batch modes. Accurate partial least squares models based on the acquired spectral data over such different cultivation conditions were built, yielding a R2 = 0.97 for biomass and plasmid productions and a RMSEP of 0.34 and 7.52, respectively; a R2 of 0.93 and a RMSEP of 0.46 and 0.33 was obtained for glucose and glycerol, respectively; the acetate model produced a R2 of 0.96 and a RMSEP of 0.32. © 2013 IEEE.