The separation and determination of chlorogenic acid inEucommia ulmoideswas studied using reversed electroosmotic flow capillary electrophoresis. The addition of 0.2 mmol/L of cetyltrimethylammonium bromide (CTAB) could change the direction of electroosmotic flow. When 10–20%(v/v) methanol was added to KH2PO4-Na2B4O7buffer at pH 8.6, the separation of chlorogenic acid from other components inEucommia ulmoidesleaf and its cortex cell culture was achieved using -20KV voltage. The effect of pH and concentration of the buffers, applied voltage and loading time on the separation and determination were also investigated. Using this method, the analysis of chlorogenic acid could be completed within 4 minutes.