Bedside diagnostics using protein biomarkers requires rapid concentration, isolation and measurement of these specific biomarkers from a complex matrix in a low cost and portable manner. Traditional separation using gel electrophoresis separates the sample into all its constituent elements and requires high voltages and/or long runtime which is often unnecessary for diagnostics where only a specific biomarker needs to be quantified. Digital isoelectric trapping can be used to isolate and concentrate proteins but requires UV cured immobiline gels that have defined pH values and are not tunable to target specific proteins. Here, we have developed miniaturized isoelectric gates to separate, concentrate and quantify a targeted biomarker based on its isoelectric point, from a complex matrix in under 20 min. We designed specific isoelectric gates to concentrate and quantify bovine serum albumin (BSA) at a concentration of 1−5 mg/mL with a peak concentration of over 300 mg/mL, while maintaining its solubility. Next, we have demonstrated isolation of human protein C from a mixture with 1000-fold higher concentration of BSA with no additional processing. Finally, we have demonstrated rapid (< 20 min) separation, concentration and quantification of ovomucoid in a fresh hen egg using dual isoelectric gates at pH 3.9 and 4.3 to trap ovomucoid with an isoelectric point of 4.1 using a low applied voltage of only 15 V. By using low cost agarose gels instead of expensive immobilines, this device reduced the cost and complexity of separation. The combination of low-cost, tunability, fast-runtime, low-operating voltage makes this technique attractive for use in point-of-care biomarker detection without specific antibody tags.