With an increased number of applications in the field of the avidin-biotin technology, the resulting demand for highly-purified protein avidin has drawn our attention to the purification process of avidin that naturally occurs in chicken egg white. The high-throughput process development (HTPD) methodology was exploited, in order to evaluate purification process alternatives to commonly used ion-exchange chromatography. In a high-throughput format, process parameters for aqueous two-phase extraction, selective precipitation with salts and polyethylene glycol, and hydrophobic interaction and mixed-mode column chromatography experiments were performed. The HTPD strategy was complemented by a high-throughput tandem high-performance liquid chromatography assay for protein quantification. Suitable conditions for the separation of avidin from the major impurities ovalbumin, ovomucoid, ovotransferrin, and lysozyme were identified in the screening experiments. By combination of polyethylene glycol precipitation with subsequent resolubilization and separation in a polyethylene glycol/sulfate/sodium chloride two-phase system an avidin purity of 77% was obtained with a yield >90% while at the same time achieving a significant reduction of the process volume. The two-phase extraction and precipitation results were largely confirmed in larger scale with scale-up factors of 230 and 133, respectively. Seamless processing of the avidin enriched bottom phase was found feasible by using mixed-mode chromatography. By gradient elution a final avidin purity of at least 97% and yield >90% was obtained in the elution pool. The presented identification of a new and beneficial alternative for the purification of the high value protein thus represents a successful implementation of HTPD for an industrially relevant purification task. (c) 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:957-973, 2015