In the present study, the special shake flasks, so-called ventilation flasks, are equipped with oxygen sensors and then an unsteady-state gas transfer model for shake flasks was developed and experimentally investigated for a wide range of gas transfer resistances (kplug). For the validation of our unsteady-state model to simulate the gas transfer in a biological system in the ventilation flasks, a strain of Corenobacterium glutamicum DM1730 was used as a model organism. For further easy processing, the resulting total mass-transfer resistance (kplug) is described as a function of the mass flow through the sterile plug (OTRplug) by an empirical equation. This equation is introduced into a simulation model that calculates the gas partial pressures in the headspace of the flask. Additionally, the gas transfer rates through the sterile closure and gas/liquid interface inside the flask are provided. This unsteady-state model would be a very useful method for scaling up from a shake flask to a fermentor; comparing the results of the gas concentration in the gas phase, there is good agreement between the introduced unsteady-state model and experimental results for the biological system.