In the present work, a series of N-(2-hydroxydodecyl)-α-amino acid amphiphiles with glycine (C12HGly), L-alanine (C12HAla), L-valine (C12HVal), L-leucine (C12HLeu), L-isoleucine (C12HIle), L-serine (C12HSer), L-threonine (C12HThr), L-asparagine (C12HAsn), L-glutamine (C12HGln), L-phenylalanine (C12HPhe), L-tryptophan (C12HTrp), L-histidine (C12HHis), L-aspartic acid (C12HAsp), and L-glutamic acid (C12HGlu) as head-group was synthesized. In order to investigate the effects of hydrocarbon chain length on the aggregation behavior, a number of CnHAla, and CnHVal amphiphiles (n = 8, 10, 12, 14, and 16) were also synthesized. The chemical structures were identified by different spectroscopic methods. Beside chemical identification of the amphiphilic molecules by conventional spectroscopic methods a number of experimental techniques, such as surface tension, fluorescence and FT-IR spectroscopy, dynamic light scattering (DLS), small angle x-ray diffraction, rheology, transmission electron microscopy (TEM), and field emission scanning electron microscopy (FE-SEM) have been used for the characterization of the amphiphiles. Surface tension measurements were performed in study the surface activity and interfacial properties at the air/water interface. Fluorescence probe technique was employed to investigate the microenvironments of the aggregates formed by the amphiphiles in aqueous solutions. The size and size distribution of the aggregates formed was studied by DLS method. Rheological measurements were performed to study the mechanical strength of the gels formed. On the other hand, microstructures of the self-assemblies formed by the amphiphiles were investigated by use of the microscopic techniques, such as TEM and FE-SEM. The chiral selectivity of selected amphiphiles was examined through enantiomeric separations of some organic racemates by micellar electrokinetic chromatography (MEKC). The chiral selectivity has been discussed in light of the aggregation behavior of amphiphiles in separation buffers.