The chemical decomposition of glutamine to ammonia and pyrrolidonecarboxylic acid was studied at 37°C in a pH range of 6.8–7.8 in different media preparations containing various amounts of fetal bovine serum. The media type influenced the decomposition rate, and the first‐order rate constants increased with increasing pH values. The serum concentration had little or no effect on the decomposition rate. The importance of chemical decomposition of glutamine on the analysis of glutamine and ammonia metabolism was illustrated by an example of batch cultivation of a hybridoma cell line. The difference between the apparent uptake rate of glutamine and the actual uptake rate (which is corrected for the chemical decomposition) is shown to be as high as 200%. Similar discrepancy between the apparent and actual ammonia production rate is observed. Mathematical analysis was carried out to develop the relationship between the apparent and actual glutamine uptake and ammonia production rates. The analysis reveals that there are three important dimensionless parameter ratios that govern the difference between the apparent and actual glutamine uptake and ammonia production rates.