The high performance liquid chromatography-electrospray ionization-mass spectrometry method has been applied to the measurement of bile acid transport activities in membrane vesicles obtained from a human bile salt export pump expressing Sf9 cells. The amounts of bile acids transported using the human bile salt export pump expressing Sf9 cells were determined using liquid chromatography-electrospray ionization-mass spectrometry method and the values of the kinetic parameters were determined to be comparable with those obtained using radioisotope-labeled substrates. The developed method was highly useful for the measurements of bile acid transport activities.