Résumé : The monitoring and optimization of hybridoma cell fed-batch cultures depend on the availability of appropriate on-line sensors for the main culture components. A simple and efficient approach to maintain hybridoma cultures in the optimal operating conditions is to regulate the substrate concentrations at the critical values (G=Gcrit and/or Gn=Gncrit) such as to control the hybridoma cells at the critical metabolism state. However, reliable glucose and glutamine probes are currently rare and/or very expensive on the market and it is necessary to design software sensors which are at same time cheap and reliable and that can be used for online measurement. In this study, the overflow metabolism model is used to develop an extended Kalman filter for online estimation of glucose and glutamine in hybridoma cell fed-batch cultures based on the considered available measurements (biomasses (on-line), lactate and ammonia (on-line or off-line)). The observability conditions are examined, and the performances are analysed with simulations of hybridoma cell fed-batch cultures. Glutamine estimation sensitivity is enforced by minimizing a cost function combining a usual least-squares criterion with a state estimation sensitivity criterion.