We have developed a visual microwell plate assay for rapid, high-throughput screening for membrane-disrupting molecules such as de novo designed pore formers, antibiotic peptides, bacterial toxins, and lipases. The detectability is based on the strong fluorescence emission of the lanthanide metal terbium(III) (Tb 3؉ ) when it interacts with the aromatic chelator dipicolinic acid (DPA). While Tb 3؉ is not strongly fluorescent alone, the binary complex emits bright green fluorescence when irradiated with uv light. For the microwell plate assay, we prepared unilamellar phospholipid vesicles that had either Tb 3؉ or DPA entrapped and the opposite molecule in the external solution. Disruption of the membranes allows the Tb 3؉ /DPA complex to form, giving rise to a visibly fluorescent solution. In plates with 20-l wells, the lower limit of visual detectability of the Tb 3؉ / DPA complex in solution was about 2.5 M. The lower limit of detectability using vesicles with entrapped Tb 3؉ or DPA was about 50 M phospholipid. We show that the membrane-disrupting effect of as little as 0.25 M or 5 pmol of the pore-forming, antibiotic peptide alamethicin can be detected visually with this system. This sensitive, high-throughput assay is readily automatable and makes possible the visual screening of combinatorial peptide libraries for members that permeabilize lipid bilayer membranes.