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Pardaxin is a fish toxin belonging to the alpha-helical, pore-forming peptide family, used in toxicological and biophysical research to study toxin-cell and -lipid-artificial membranes interactions. We investigated the membrane interaction of two pardaxin analogues using a colorimetric phospholipid/polydiacetylene biomimetic assay. In this assay, polydiacetylene undergoes visible, concentration dependent, blue-red transformation induced through interactions of pardaxins with the vesicle membrane. Pardaxins P4 and P5, are composed of 33 amino acids, but differ in a single amino acid substitution at the carboxy-terminal (G31 to D31, respectively) known to decrease the pore forming activity. Addition of pardaxins in the colorimetric assay induced dose-dependent color transitions with different kinetics. The colorimetric analysis could distinguish between different pardaxins-membrane interaction profiles, suggesting bilayer surface association for P4 and vesicle membrane penetration for P5. The colorimetric assay could distinguish between pardaxins membrane interaction profiles although circular dichroism spectra of vesicle-interacting pardaxins did not indicate a significant difference in the secondary structure between these two toxin analogues. The colorimetric platform utilized in the present report represents a useful assay with general applications for studying membrane interactions of peptides in general and pore-forming toxins in particular, and may become an important tool for evaluating quantitative toxin structure-activity relationship.