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A mixture of the cationic target analyte and the terminating electrolyte (TE) from the two sample reservoirs was injected under FESI conditions within the two sample\u2010loading channels. At the double T\u2010junction, the stacked analyte zones were further concentrated under field\u2010amplified stacking conditions and then subsequently focused by transient\u2010isotachophoresis and separated along the separation channels. The proposed multi\u2010stacking strategy was verified under a Universal Serial Bus (USB) fluorescence microscope employing Rhodamine 6G as the model analyte. This developed approach was subsequently used to monitor the target quinine present in human plasma samples. The total analysis time for quinine was approximately 200\u00a0s with a sensitivity enhancement factor of approximately 61 when compared to the typical gated injection. The detection and quantification limits of the developed approach for quinine were 3.0\u00a0\u03bcg\/mL and 10\u00a0\u03bcg\/mL, respectively, with intraday and interday repeatability (%RSDs, <jats:italic>n<\/jats:italic> = 5) of 3.6 and 4.4%. 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