Transcutaneous (TCI) immunization is a novel vaccination approach that provides many advantages over traditional parenteral vaccination. However, a major barrier to TCI is mediating penetration of vaccine antigens through the stratum corneum (SC) to the deeper tissue layers. Many approaches have been investigated for enhancing drug penetration into the skin including microneedles (MNs) to transiently breach the SC barrier and incorporation of vaccines into penetration-enhancing delivery systems. This study utilized MNs and a lipid-based colloidal delivery system (cubosomes) as a synergistic approach for the delivery of vaccines through the skin. The penetration and permeation of the peptide antigen into and through skin were analysed quantitatively and qualitatively using techniques including Optical Coherence Tomography and two-photon microscopy. It was found that while the use of MNs increased the permeation of an aqueous peptide mixture through skin, cubosome-formulated peptide and cubosomes were retained in the skin. Therefore, it is proposed that a combined approach using MNs and cubosomes will be an efficient system for the local delivery of antigen to immunocompetent cells in the skin.