Electromembrane extraction (EME) coupled with high performance liquid chromatography and ultraviolet detection was developed for determination of amphetamine-type stimulants in human urine samples. Amphetamines migrated from 3mL of different human urine matrices, through a thin layer of 2-nitrophenyl octyl ether (NPOE) containing 15% tris-(2-ethylhexyl) phosphate (TEHP) immobilized in the pores of a porous hollow fiber, and into a 15μL acidic aqueous acceptor solution present inside the lumen of the fiber. Equilibrium extraction conditions were obtained after 7min of operation. Experimental design and response surface methodology (RSM) were used for optimization of EME parameters. Under optimal conditions, amphetamines were effectively extracted with recoveries in the range of 54–70%, which corresponded to preconcentration factors in the range of 108–140. The calibration curves were investigated in the range of 0–7μgmL −1 and good linearity was achieved with a coefficient of estimation better than 0.991. Detection limits and inter-day precision (n=3) were less than 0.01μgmL −1 and 11.2%, respectively.