Pleckstrin homology (PH) domains are present in key proteins involved in many vital cell processes. For example, the PH domain of Bruton’s tyrosine kinase (Btk) binds to phosphatidylinositol triphosphate (PIP₃) in the plasma membrane after stimulation of the B-cell receptor in B cells. Mutations in the Btk PH domain result in changes in its affinity for PIP₃, with higher binding leading to cell transformation in vitro and lower binding leading to antibody deficiencies in both humans and mice. We describe here a fluorescence resonance energy transfer (FRET)-based biochemical assay that directly monitors the interaction of a PH domain with PIP₃ at a membrane surface. We overexpressed a fusion protein consisting of an enhanced green fluorescent protein (GFP) and the N-terminal 170 amino acids of a Tec family kinase that contains its PH domain (PH170). Homogeneous unilamellar vesicles were made that contained PIP₃ and octadecylrhodamine (OR), a lipophilic FRET acceptor for GFP. After optimization of both protein and vesicle components, we found that binding of the GFP-PH170 protein to PIP3 in vesicles that contain OR results in about a 90% reduction of GFP fluorescence. Using this assay to screen 1440 compounds, we identified three that efficiently inhibited binding of GFP-PH170 to PIP₃ in vesicles. This biochemical assay readily miniaturized to 1.8-μl reaction volumes and was validated in a 3456-well screening format.