In the current study, a new method is introduced with which the rate-limiting factor of biodegradation processes of hydrophobic chemicals in organic and aqueous systems can be determined. The novelty of this approach lies in the combination of a free concentration-based kinetic model with measurements of both free and total concentrations in time. This model includes microbial biodegradation activity of the chemical in the aqueous phase and chemical sorption kinetics with respectto organic carbon and aqueous phases. The time dependency of free and total concentrations of 7-acetyl-1,1,3,4,4,6-hexamethyltetrahydronaphthalene and 7-acetyl-1,1,3,4,4,6-hexahydro-4,6,6,7,8,8-hexamethylcyclopenta(g) -2-benzopyrane in activated sludge was experimentally determined in vitro. Evaporation losses from the test system were also determined. Least-squares regression to optimize the model parameters resulted in a model that is in accordance with the experimental data. Additionally, the model shows that a comparison between the decrease of free and total chemical concentrations in time, in combination with an independent measurement of the organic carbon/water partition coefficient provides information about the rate-limiting step of the degradation process. This information can be used by sewage treatment plant managers to decide whether the microbial biodegradation activity itself or the desorption from organic carbon to the aqueous phase should be improved.