A convenient and reliable liquid chromatographic method with electrochemical detection was developed for the simultaneous determination of reduced coenzyme Q(ubiquinol), oxidized coenzyme Q(ubiquinone) and alpha-tocopherol in various tissues. The compounds of interest were separated on a reverse-phase C18 column under the mobile phase of 7 g/L NaClO4 in ethanol-methanol-70% HClO4 (800:200:1, volume ratio) and detected simultaneously by using a series of detection cells. Electrochemical guard cell (upstream of the injector) was used for eliminating interference for mobile phase. Ubiquinones were reduced in the -550 mV conditioning cell (downstream of the column). The resultant ubiquinols and original ubiquinols and alpha-tocopherol existing in the sample were easily oxidized in the 150 mV analytical cell. The linear range of the compounds were from 5 micrograms/L to 200 micrograms/L with correlation coefficients from 0.9976 to 0.9999. The detection limits were from 0.23 microgram/L to 0.68 microgram/L. The method was applied satisfactorily to the simultaneous determination of the amounts of ubiquinol-9, ubiquinol-10, ubiquinone-9, ubiquinone-10 and alpha-tocopherol in mice plasma and tissue mitochondria.