Comprehension of the mechanisms of hydrophobic interaction chromatography is not as deep as for other chromatographic steps. This article shows the combination of modeling and experimental model parameter determination. Original mammalian cell culture is applied for validation instead of often-used test systems with for example, three pure proteins. This approach can have a huge benefit for process development and optimization of production processes under project conditions. To simulate the separation by hydrophobic interaction chromatography, a distributed plug flow model with inner pore diffusion is applied. The needed model parameters are determined by small scale experiments on a 1ml column to reduce feed amount consumption and experimental procedure. Effects caused by system setup are optimized in relation to the volume. Gradient experiments are performed for the determination of isotherm parameters. The model parameters determined are experimentally validated at different set-ups of operation parameters as well as scaled up to 10ml columns. The practicability of the proposed stepwise procedure of physico-chemical modeling in combination with laboratory scale experiments for the prediction of separation performance of original mammalian cell culture is proved. A model parameter determination directly with fermentation mixture is as well feasible.