AbstractThe use of in‐situ near infrared spectroscopy (NIRS) as a tool for monitoring four key analytes in a CHO‐K1 animal cell culture was investigated. Previous work using on‐line NIRS to monitor bioprocesses has involved its application ex‐situ where the analyzer is physically outside the fermentor, or to microbial bioprocesses. This novel application of NIRS to monitor analytes within an animal cell culture using a steam sterilizable in‐situ fiber optic probe is very important for furthering the use of NIRS within the bioprocessing industry. The method of calibration used to develop the models involved the use of large data sets so that all likely variation in stoichiometry was incorporated within the models. Successful models for glucose, lactate, glutamine, and ammonia were built with Standard Error of Predictions (SEP's) of 0.072 (g/L), 0.0144 (g/L), 0.308 (mM), and 0.036 (mM), respectively of the total concentration range. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 84: 13–19, 2003.