For the evaluating of the absorption, tumor affinity and metabolism of wilfoside C3N and wilfoside C1N (two main C21 steroidal glycosides from Baishouwu), an LC-MS/MS method was developed. Plasma or tumor homogenate samples were extracted by liquid-liquid extraction with ethyl acetate after internal standard (ginsenoside Rh2) spiked. The separation was performed by a Luna C18 column (3.0 microm, 2.0 mm x 50 mm) with gradient elution. The method was fully validated and successfully applied to determine the concentrations of the parent drugs and metabolites after intragastric administration of wilfoside C3N and wilfoside C1N to mice respectively.