Due to lack of robust detection method, the application of inside-out vesicle in characterizing drug interaction with ABC transporter has been largely limited to radiolabeled chemicals or inhibitory screening assay. The aim of this study is to develop a robust detection method using liquid chromatography-Tandem mass spectrometry for characterizing drug interaction with two major ABC transporters using vesicle uptake assay, e.g. human MRP2 and human BCRP. Inside-out vesicles were prepared from insect Sf9 cells infected with baculovirus expressing hMRP2 and hBCRP. The MRP2 uptake of radiolabeled chemicals or non-radiolabeled counterparts: 3H- leukotriene C4 or LTC4 and 3H-estradiol-17β-glucuronide or E17βG, BCRP uptake of 3H-estrone-3-sulfate or E3S and 3H-methotrexate or MTX were characterized using a rapid filtration system as described previously. For non-radiolabeled chemicals, the dried glass fiber filter plate is extracted with 200 μL of elution buffer. The extracts were eluted into a 96-well receiver plate by centrifugation at 4000 rpm for 5 min, which were subjected to LC-MS/MS quantification. Kinetic assay (Km and Vmax) and IC50 inhibition assays for benzbromarone on MRP2 uptake activity, Sulfasalazine on BCRP uptake activity were conducted using both radiolabeled and non-radiolabeled substrates. The kinetic parameters of selected MRP2 substrates and BCRP substrates and IC50 of selected MRP2 and BCRP inhibitor generated using LC-MS/MS method is comparable to those generated using radiolabeled chemicals. No significant difference was observed for the solvent based elution buffer compared with aqueous based elution buffer for both kinetic assay and inhibitory assay. In this study, the assay method was developed for inside-out vesicle uptake assay for non-radiolabeled chemicals using rapid filtration system and LC-MS/MS detection approach. The method was validated using selected probe substrates for both human MRP2 and BCRP transporters.