Human ABCG2 (breast cancer resistance protein, BCRP) is an important efflux transporter which exhibits broad substrate specificity and which is found in many tissues. The purpose of the present study was to develop a 96-Transwell assay using an MDCK II cell line stably transfected with ABCG2 (MDCK II/ABCG2) to identify ABCG2 substrates. In this assay, which also incorporates a high throughput mass spectrometry method for quantification, efflux activity of the MDCK II/ABCG2 cells was evaluated by monitoring the basolateral-to-apical/ apical-to-basolateral (B to A/A to B) efflux ratio of several substrates. Mean MDCK II/ABGC2 efflux ratios for 2 microM prazosin, SN-38, and Cl 033 were 2.8, 7.6, and 2.4, respectively, and the mean efflux ratio for 10 microM mitoxantrone was 5.0. Interday variability of the assay was low (CV = 10-29% for control compounds at 2 microM). Our data indicate that a compound tested at 2 microM can be considered a substrate of ABCG2 if its ratio of ratios (MDCK II/ABCG2 efflux ratio)/ (MDCK II efflux ratio) is > 1.2. This assay provides an efficient, high throughput means to identify ABCG2 substrates in drug discovery.