Cationic antimicrobial peptides (AMPs) are present in many organisms and can kill a broad range of microorganisms by membrane permeabilization. Consequently short cationic peptides may serve as a new generation of antibiotics, especially in light of the alarming increase in microbial resistance to the conventional antibiotics. Membrane pore-forming peptides and known AMPs share a similar mechanism of action in lipid bilayers in vitro. In an effort to develop a novel strategy to screen for new peptide antibiotics, we used combinatorial chemistry and high-throughput screening. We selected potent pore-forming peptides from a combinatorial library of 16,000 putative short, cationic amphipathic beta-strand peptides of 9-15 residues using our screening assay. We selected 10 water-soluble potent pore-forming peptides, less than 0.1% of library population. These pore-forming peptides were soluble in buffer but bind strongly to lipid vesicles and induce rapid transient leakage of encapsulated contents. All of the peptides were beta-sheets in membranes. We found that these peptides release the contents of liposomes in all-or-none leakage mechanism. These pore-forming peptides have potent, broad-spectrum antimicrobial activity ranging from 2 to 4 μM minimum sterilization concentrations (MSC). But they have little lytic activity against erythrocytes or living human cells. In an attempt to select AMPs directly from the library, we developed a bioassay, and compared the results with the liposome-based screening assay. We found that the bioassay may allow us to screen for species-specific peptides while the vesicle-based assay gives broad-spectrum AMPs. Here, we propose that a rational combinatorial design coupled with a function-based screening assay is a powerful method to select peptides that are not only potent membrane-active, but also broad-spectrum antibiotics.