L-Glutamine (L-Gln) instability in liquid media is a well-known fact. Also, negative effect of ammonia, one of the L-Gln degradation products, on viability of many cell cultures and on replication of different viruses has been described. However, negative effects of ammonia have been reported in doses excessively exceeding those that could be generated in regularly used liquid culture media due to spontaneous L-Gln breakdown (below 2 mM). Traditional virus vaccine production processes have been established and registered involving L-Gln containing media use. Eventual culture media replacement in the regular production process belongs to the major regulative changes that require substantial financial expenses. The aim of this study was to evaluate the effect of storage of Minimum Essential Media with Hanks salts on their relevant biological functions during virus vaccine production process in relation to L-Gln decrease. Our results show a cell type dependent effect of spontaneous L-Gln degradation during medium storage. They also suggest that for cell cultures used in measles, mumps, and rubella virus production the media retain their functionality in respect to cell viability or virus growth over a certain time window despite L-Gln degradation.