The alignment of membrane proteins provides pertinent structural and dynamic information. Structural topology data gleaned from such studies can be used to determine the functional mechanisms associated with a wide variety of integral membrane proteins. In this communication, we successfully demonstrate, for the first time, the determination of the structural topology and helical tilt of an antimicrobial peptide magainin 2 using aligned X-band spin-label EPR spectroscopic techniques. This novel comparison unlocks many possibilities utilizing EPR spectroscopy to probe antimicrobial peptide topologies with increased sensitivity and may also give further clues to elucidate their corresponding mechanisms.