Chiral cis/trans diastereomeric cationic amphiphiles have been synthesized and studied. They are based on β-amino acids and contain C12- and C16-alkyl chains, respectively, as hydrophobic tails while the polar head consists of an ammonium cation linked to a cyclobutane ring. Their physicochemical properties, such as the cmc (critical micellar concentration), the pKa, the ratio of cationic versus non-ionic species, and the surface tension are strongly dependent on the pH of the medium. At the same time the aggregation state influences on the apparent pKa values of the aggregates, with release of protons at the cmc whose values, as well as those of the adsorption effectiveness, account for their efficient surfactant behavior. A tail-length effect is manifest because surfactant cmc of compounds with a C16-alkyl chain are smaller than the C12-ones, although not as small as expected. On the other hand, while for C12-surfactants the role of the stereochemistry on the physicochemical parameters is patent, it is not very clear for C16-surfactants. The stereochemistry also determines the predominant mode of self-assembly since the trend for cis-isomers is to form micelles or vesicles while trans-isomers preferably form fibers. CD spectroscopy confirmed the aggregation through the formation of intermolecular hydrogen bonds between the amide groups of the monomers. An alternative method to determine the cmc of these surfactants is provided by considering the relationship of the CD λmax with concentration, although it is restricted to those surfactants in which the chromophore is located in a chiral environment. Furthermore, the non- toxicity of the surfactants has been verified by the MTT assay. This characteristic, jointly with the efficiency and the good properties shown, the fact that the cationic species are present in a high concentration at physiological pH, as well as the weak acid behavior of the aggregates, confirm these amphiphiles as promising candidates to be used as non-viral vectors for gene therapy applications.