Large collections of purified proteins have become essential to systems biology programs for generating protein ligands and for validating protein interactions. These large protein collections will likewise benefit structural studies, and quests for protein-based therapeutics. Whether many protein targets need to be produced, such as a microbial proteome, or a few poorly expressing protein targets need to be expressed as soluble fragments (divide and conquer approach), efficient high-throughput processing can be a bottleneck (1). This chapter describes a method for efficient high-throughput purification of hexahistidine-tagged proteins that are expressed in Eschericia coli (E. coli) using immobilized metal affinity chromatography (IMAC) (2) in a 96-well format. This approach is particularly suitable for proteomic applications that require modest amounts of highly purified proteins to be generated very efficiently. This approach is also very useful for identifying protein targets that are most amenable to scaled-up production for use in structural studies. The typical yield of proteins purified using this system is 50 150 micrograms, which is generally greater than that of many in vitro expression systems and much less costly. The method as described has been optimized for purifying approximately 150 micrograms of hexahistidine-tagged protein, but the method is flexible so that the amount of affinity matrix and culture volumes can be adjusted for optimal binding capacity and consequently highest purity. Although the method detailed here uses IMAC to purify hexahistidine-tagged proteins, this basic platform can be used with many other tags and affinity resins.