Lysis of Gram-negative bacteria by dsDNA phages is accomplished through either the canonical holin-endolysin pathway or the pinholin-SAR endolysin pathway. During lysis, the outer membrane (OM) is disrupted, typically by two-component spanins or unimolecular spanins. However, in the absence of spanins, phages use alternative proteins called Disruptin to disrupt the OM. The Disruptin family includes the cationic antimicrobial peptide gp28, which is found in the virulent podophage φKT. In this study, EPR spectroscopy was used to analyze the dynamics and topology of gp28 incorporated into a lipid bilayer, revealing differences in mobility, depth parameter, and membrane interaction among different segments and residues of the protein. Our results indicate that multiple points of helix 2 and helix 3 interact with the phospholipid membrane, while others are solvent-exposed, suggesting that gp28 is a surface-bound peptide. The CW-EPR power saturation data and helical wheel analysis confirmed the amphipathic-helical structure of gp28. Additionally, course-grain molecular dynamics simulations were further used to develop the structural model of the gp28 peptide associated with the lipid bilayers. Based on the data obtained in this study, we propose a structural topology model for gp28 with respect to the membrane. This work provides important insights into the structural and dynamic properties of gp28 incorporated into a lipid bilayer environment.