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This paper reports a dual-column miniseparator that depletes and separates target analytes from a mixed sample. The prototype was designed to address the need for a sample preparation stage in portable point-of-care testing devices, and its efficacy was tested with Immunoglobulin G (IgG), α-fetoProtein (AFP), and fibrinogen. The miniseparator connects two minicolumns in series: The first column is an affinity minicolumn containing Protein A/G agarose beads, and the second column is a size exclusion chromatography (SEC) minicolumn that separates mixed analytes by size. When a mixture of IgG, fibrinogen, and AFP flows through the dual-column miniseparator, IgG is depleted in the first column, and AFP is separated from fibrinogen in the second column. Each protein was conjugated with fluorescent dyes, and IgG depletion and SEC separation results were confirmed by evaluating the eluents' fluorescent intensity. Red (IgG), green (AFP), and blue (fibrinogen) intensities showed that, in 10 min, the first column (affinity minicolumn) depleted over 99.5% of IgG, and the second column (SEC minicolumn) separated AFP (1 μg/mL ) from a fibrinogen-rich solution (10 μg/mL) with 1.76 resolution and 0.093-mm plate height.