Gradient elution chromatography plays a very important role in the large-scale separation of biomolecules. The major problem with gradient elution is that the development, scaleup, and optimization of the process depend on trial and error. This is usually time-consuming and expensive, especially for large-scale processes. Using chemical engineering concepts, the optimization of gradient elution chromatography consisted of two parts. In the first part, rate parameters and gradient correlations were determined according to experimental data from isocratic elution runs. In the second part, which is the main focus of this study, the optimization strategy of gradient elution chromatography based on the resolution optimization factor was developed. The resolution optimization factor considered not only the product purity and yield but also the operating time and product dilution. The methodology of optimizing gradient profiles for elution chromatography was demonstrated for protein purification.