The organization of bilayers depends on the physicochemical and structural properties of the molecules that compose the membrane. The phospholipid molecular shape depends on the relative volumes of their polar headgroups and non-polar acyl chains. In this work, we study the effect of DHPC concentration over the bilayer of large unilamellar vesicles (LUV) prepared with different proportions of dipalmitoylphosphatidylcholine / sphingomyelin / dihexanoylphosphatidylcholine (DPPC/SM/DHPC) using fluorescence spectroscopy and molecular dynamics (MD) simulations. In this work, we use Laurdan´s generalized polarization (GP) and 1,6-diphenyl-1,3,5-hexatriene (DPH) fluorescence anisotropy to observe the effect of DHPC concentration on dynamic properties of both, the interface and hydrophobic region. Detailed information, at atomistic scale, was obtained by trajectory calculations of accessible solvent area (ASA), partial densities and order parameters using Molecular Dynamics (MD). The results indicate that DHPC induces global packing defects in the membrane, favoring the liquid crystalline state for the system with higher concentration of DHPC.