Bacopaside I is a triterpenoid saponin, a major compound in Bacopa monnieri, which exhibits several potentially useful clinical activities including memory enhancement. Accordingly, a sensitive liquid chromatography tandem mass spectrometric method was developed and validated to determine bacopaside I in rat serum, urine, and feces. Digitoxin was the internal standard. Simple protein precipitation was used in the sample preparation. The separation was performed using a C18 column with gradient elution, and electrospray ionization in the negative ion mode was selected. The method proved to be precise (both intra- and inter-day CV <13.2% in serum, urine, and feces), accurate (intra- and inter-day recovery within 93.1–112.0 %), and selective, while providing high sensitivity (LLODs were 0.13 ng for rat serum and urine and 0.25 ng for rat feces and LLOQs were 0.13 ng for rat serum and urine and 0.25 ng for rat feces). We successfully applied this method to the pharmacokinetics of bacopaside I in rats. This report describes mean residence time and elimination of bacopaside I.