Résumé : In order to mantain hybridoma cell cultures in optimal operating conditions, on-line measurements of glutamine and glucose concentrations are required, implying the availability of probes, which are expensive and with poor durability. A way to overcome this problem is to design software sensors. In this work, both Extended and Unscented Kalman Filters are developed in order to estimate glucose and glutamine concentrations, based on biomass, lactate and ammonia on-line measurements. System observability conditions are first examined. The performances of both software sensors are analyzed with simulations of hybridoma cell cultures in fed-batch and continuous bioreactor operating modes. Three different tests are conducted in order to compare the performance of both observers: continuous culture with constant feeding profile, fed-batch culture with both constant and exponential feeding profiles. Also, two different sets of parameters are investigated: the ones obtained by using the least-squares method in order to minimize the error between model predictions and experimental measurements, and the ones which are modified by minimizing a cost function combining the usual least-squares criterion with a state estimation sensitivity criterion.