A short-end injection CE method combining field-amplified sample stacking (FASS) is presented for the analysis of fluoxetine (FL) and norfluoxetine in plasma. In this study, FASS enhanced the sensitivity about 1100-fold, while short-end injection reduced the analysis time to less than 4 min. Parameters involved in the separations were investigated using a central composite design (CCD) and response surface methodology to optimize the separation conditions in a total of only 32 runs. Samples injected into the capillary for 99.9 s at a voltage of -5 kV were stacked in a water plug (0.5 psi, 9 s). Baseline resolution of FL and its major metabolite was achieved using a BGE formulation consisting of phosphate-triethanolamine at low pH, and a separation voltage of -10 W Five percent methanol was added as organic modifier to enhance selectivity and resolution. The linear range was between 10 and 500 ng/mL (r > 0.9946), covering the expected plasma therapeutic ranges. The LOD in plasma were 4 ng/mL (S/N = 3), a value comparable to that obtained using LCMS, showing the success of the on-line stacking technique. Our method was also successfully validated in quantification and pharmacokinetic studies with three volunteer plasma samples and could be applied to pharmacogenetic studies.