In the biopharmaceutical industry, process development has to overcome the paradox between increasing demands on product quality and aggressive overall development time lines. Streamlining development and scale-up has become a requirement in order to cope with these constraints. Hydrophobic interaction chromatography (HIC) has gained popularity as an efficient tool in downstream process development, which represents an orthogonal method compared to other widely used chromatography principles. Thus, host cell protein (HCP) removal combined with the separation of product variants based on subtle differences in hydrophobicity are major strengths of this separation technique. ne development and scale-up of a preparative HIC step for a therapeutic protein for human use is described. Issues like the removal of product- and process-related impurities have been addressed. The direct transferability of the results, from scale-down optimization experiments via the pilot scale to the final production scale, clearly show the robustness and power of HIC in this process context.