Positron annihilation lifetime spectroscopy (APLS) has potential as a novel rapid characterisation method for self-assembly amphiphile systems; however, lack of systematic correlation of PALS parameters with structural attributes has limited its more widespread application. In this study, using the well-characterized phytantriol/water and phytantriol/vitamin E acetate/water self-assembly amphiphile systems, the impact of systematic structural changes controlled by changes in composition and temperature on PALS parameters has been studied. The PALS parameters (orthopositronium (oPs) lifetime and intensity signatures) were shown to be sensitive to the molecular packing and mobility of the self-assembled lipid molecules in various lyotropic liquid crystalline phases, enabling differentiation between liquid crystalline structures. The oPs lifetime, related to the molecular packing and mobility, is correlated with rheological properties of the individual mesophases. The oPs lifetime links the lipid chain packing and mobility in the various mesophases to resultant macroscopic properties, such as permeability, which is critical for the use of theses mesophase structures as diffusion-controlled release matrices for active liposoluble compounds.