Objective To develop an ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method to quantify the uptake of panaxynol in cultured Caco-2 cells. Methods The chromatographic separation was performed on Acquity BEH C18 column (2.1 mm×100 mm, 1.7 μm) with aqueous methanol as the mobile phase, using gradient elution at 0.3 mL/min. A triple-quadruple mass spectrometer employing electrospray ionization in positive ion mode was developed, and panaxynol in Caco-2 cells was determined using multiple reaction monitoring of precursor→product ion transitions at m/z 227→129 for quantification and m/z 227→143 for confirmation. Results The established method was validated by determining the linearity (r2>0.99), precision (≤6.2%) and accuracy (－6.7% to 2.1%). The limit of detection for panaxynol was 4 ng/mL. When incubated for 2 h at 37 ℃, the uptake was (20.7±1.8) nmol/mg protein for 50 μmol/L panaxynol and (21.8±1.7) nmol/mg protein for 100 μmol/L panaxynol, and there was no significant difference between them （P>0.05）. Conclusion This method is sensitive, reliable and specific, and can be used in determination of panaxynol in Caco-2 cells.