To study the pharmacokinetics of Chan Su, a sensitive and selective method was developed and validated for the determination of five main bufadienolides (cinobufagin, resibufogenin, bufalin, bufotalin and arenobufagin) in rat plasma. The analytes were extracted by liquid-liquid extraction with ethyl acetate after internal standard (IS, caudatin) spiked. The separation was performed by a ZORBAX SB-C18 column (3.5 microm, 2.1 mmx100 mm) and a C18 guard column (5 microm, 4.0 mmx2.0 mm) with an isocratic mobile phase consisted of acetonitrile-water-formic acid (50:50:0.05, v/v/v) at a flow rate of 0.3 mL/min. The Agilent G6410A triple quadrupole LC/MS system was operated under the multiple reaction monitoring mode (MRM) using the electrospray ionization technique in positive mode. The nominal retention times for cinobufagin, resibufogenin, bufalin, bufotalin, arenobufagin and caudatin were 3.07, 3.55, 2.30, 1.62, 1.22 and 3.43 min, respectively. All analytes showed good linearity in a wide concentration range (r>0.995) and their lower limits of quantification (LLOQ) were all 1.0 ng/mL. The method was linear for all analytes with correlation coefficients>0.995 for all analytes. The average extract recoveries of the five analytes from rat plasma were all over 85%, the precisions and accuracies determined were all within 15%. This method has been successfully applied to pharmacokinetic study of Chan Su in rats following oral administration.