Biopharmaceutical separations require tremendous amounts of optimization to achieve acceptable product purity. Typically, large volumes of reagents and biological materials are needed for testing different parameters, thus adding to the expense of biopharmaceutical process development. This study demonstrates a versatile and customizable microscale column (mu Col) for biopharmaceutical separations using immobilized metal affinity chromatography (IMAC) as an example application to identify key parameters. mu Cols have excellent precision, efficiency, and reproducibility, can accommodate any affinity, ion-exchange or size-exclusion-based resin and are compatible with any high-performance liquid chromatography (HPLC) system. mu Cols reduce reagent amounts, provide comparable purification performance and high-throughput, and are easy to automate compared with current conventional resin columns. We provide a detailed description of the fabrication methods, resin packing methods, and mu Col validation experiments using a conventional HPLC system. Finite element modeling using COMSOL Multiphysics was used to validate the experimental performance of the mu Cols. In this study, mu Cols were used for improving the purification achieved for granulocyte colony stimulating factor (G-CSF) expressed using a cell-free CHO in vitro translation (IVT) system and were compared to a conventional 1 ml IMAC column. Experimental data revealed comparable purity with a 10-fold reduction in the amount of buffer, resin, and purification time for the mu Cols compared with conventional columns for similar protein yields.