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Abstract Matrix components are known to significantly alter the ionization of a target analyte in ESI‐based measurements particularly when working with complex biological samples. This issue however may be alleviated by extracting the analyte of interest from the original sample into a relatively simple matrix compatible with ESI mass‐spectrometric analysis. In this article, we report a microfluidic device that enables such extraction of small peptide molecules into an ESI‐compatible solvent stream significantly improving both the sensitivity and reproducibility of the measurements. The reported device realizes this analyte extraction capability based on the free‐flow zone electrophoretic fractionation process using a set of internal electrodes placed across the width of the analysis channel. Employing lateral electric fields and separation distances of 75 V/cm and 600 µm, respectively, efficient extraction of the model peptide human angiotensin II was demonstrated allowing a reduction in its detection limit by one to three orders of magnitude using the ESI‐MS method. The noted result was obtained in our experiments both for a relatively simple specimen comprising DNA strands and angiotensin II as well as for human serum samples spiked with the same model peptide.