In-line monitoring tools are still required to understand and control animal cell processes, particularly in the case of vaccine production. Here, in situ near-infrared spectroscopy (NIRS) quantification of components in culture media was performed using microcarrier-based cultivations of adherent Vero cells. Because microcarriers were found to interfere with NIRS spectra acquisition, a suitable and innovative in situ calibration was developed for bioreactor cultures. A reliable and accurate NIRS technique for the quantification of glucose and lactate was established, with a calibration standard error of 0.30 and 0.21 gl(-1), respectively. The robustness of this method was evaluated by performing NIRS calibration with operating conditions similar to those of industrial processes, including parameters such as microcarrier concentrations, cell seeding states and changes in analyte concentration due to feed and harvest strategies. Based on this calibration procedure, the predicted analyte concentrations in unknown samples was measured by NIRS analyses with an accuracy of 0.36 gl(-1) for glucose and 0.29 gl(-1) for lactate. (C) 2010 Elsevier Ltd. All rights reserved.