Widespread resistance to antibiotics in current clinical use is increasing at an alarming rate. Novel approaches in antimicrobial therapy will be required in the near future to maintain control of infectious diseases. An enormous array of small cationic peptides exists in nature as part of the innate defense systems of organisms ranging from bacteria to humans. For most naturally occurring linear peptides, such as magainins and cecropins, a common feature is their capacity to form an amphipathic α-helix (with polar and nonpolar groups on opposite faces of the helix), a structural feature believed to be important in their antimicrobial function as membrane-lytic agents. A massive effort over the past two decades has resulted in a better understanding of the molecular mechanism of antimicrobial peptides and the production of more potent analogues. To date, however, few of these peptides have been shown to have clinical efficacy, especially for systemic use, in large part due to insufficient selectivity between target and host cells. Recently, we developed a new strategy in the design of antimicrobial peptides. These linear cationic peptides, which form amphipathic β-sheets rather than α-helices, demonstrated superior selectivity in binding to the lipids contained in bacterial vs. mammalian plasma membranes. Here we describe methods to evaluate the structure and function of cationic antimicrobial peptides.