Caco-2 cells, the human colon carcinoma cells, are typically used for screening compounds for their permeability characteristics and P-glycoprotein (P-gp) interaction potential during discovery and development. The P-gp inhibition of test compounds is assessed by performing bi-directional permeability studies with digoxin, a well established P-gp substrate probe. Studies performed with digoxin alone as well as digoxin in presence of test compounds as putative inhibitors constitute the P-gp inhibition assay used to assess the potential liability of discovery compounds. Radiolabeled 3 H-digoxin is commonly used in such studies followed by liquid scintillation counting. This manuscript describes the development of a sensitive, accurate, and reproducible LC–MS/MS method for analysis of digoxin and its internal standard digitoxin using an on-line extraction turbulent flow chromatography coupled to tandem mass spectrometric detection that is amendable to high throughput with use of 96-well plates. The standard curve for digoxin was linear between 10nM and 5000nM with regression coefficient (R 2 ) of 0.99. The applicability and reliability of the analysis method was evaluated by successful demonstration of efflux ratio (permeability B to A over permeability A to B) greater than 10 for digoxin in Caco-2 cells. Additional evaluations were performed on 13 marketed compounds by conducting inhibition studies in Caco-2 cells using classical P-gp inhibitors (ketoconazole, cyclosporin, verapamil, quinidine, saquinavir etc.) and comparing the results to historical data with 3 H-digoxin studies. Similarly, P-gp inhibition studies with LC–MS/MS analytical method for digoxin were also performed for 21 additional test compounds classified as negative, moderate, and potent P-gp inhibitors spanning multiple chemo types and results compared with the historical P-gp inhibition data from the 3 H-digoxin studies. A very good correlation coefficient (R 2 ) of 0.89 between the results from the two analytical methods affords an attractive LC–MS/MS analytical option for labs that need to conduct the P-gp inhibition assay without using radiolabeled compounds.