Methods based on immobilized-metal affinity chromatography (IMAC) technology for the isolation of 6xHis-tagged proteins offer a one-step purification process that is both robust and meets the challenge of quantitatively purifying thousands of proteins with differing structures and characteristics. To perform this method in a high-throughput, automated format, protocols have been developed that can be run on lab automation workstations. Ready-to-run protocols covering a wide range of protein purification and assay applications, and which rely on the well-established 6xHis-Ni-NTA IMAC technology, are available. An Ni-NTA magnetic bead-based protocol allows micro-scale purification of up to 15 microg of 6xHis-tagged protein per well. If larger amounts of purified protein are needed, a vacuum-controlled Ni-NTA resin-based process provides a convenient medium-scale method for purification of up to 300 microg of 6xHis-tagged protein in a 96-well format. This protocol has been adapted to further increase the yield of 6xHis-tagged proteins allowing purification of up to several milligrams of highly pure protein per well. The protocol can process 96 samples, each derived from up to 25 mL culture volume (E. coli), in less than 3 h. Examples of purification and assay applications using the protocols mentioned above, and data on reproducibility and cross-contamination-free processing are shown.