A method was developed to determine brazilin and protosappanin B in natural products by CE after acid barrage stacking. The optimum conditions were as follows: a BGE of 20mM sodium tetraborate (pH 9.2) containing 6% v/v of methanol, hydrodynamic injection (0.5psi, 65s) followed by hydrodynamic injection of 150mM citric acid (pH2.3; 0.5psi, 22s), and separated with +25kV. Under these conditions, brazilin and protosappanin B were separated with a sample-to-sample time less than 13min and detection limits of 0.28g/mL and 0.15g/mL, respectively. The applicability of the developed method was demonstrated by the detection of brazilin and protosappanin in methanol extract of sappan lignum.