In this study the gas-phase conformer preferences of Gramicidin A (GA), a linear antimicrobial pentadecapeptide, were investigated directly from aqueous solutions of lipid vesicle bilayers using a mixing tee-electrospray ionization (MT-ESI) setup coupled with ion mobility mass spectrometry (IM-MS). The required time for GA sample preparation was decreased by approximately 50% using MT-ESI when compared to previously reported methods which required freeze-drying of samples. Using an MT-ESI approach to analyze samples of GA associated with POPC (16:0, 18:1 PC) and DEPC (22:1 PC) lipid bilayers yielded dimer conformer preferences comparable to results obtained using more lengthy protocols. GA analogues that contain leucine to lysine substitutions were analyzed; these analogues yielded more hydrophilic GA dimers owing to the hydrophilicity of lysine head groups. The conformer preferences of lipid bilayer associated hydrophilic GA analogues can be obtained owing to disassociation of lipids during the fast mixing time MT-ESI process. The data for both GA analogues associated with negatively charged POPC/POPG (16:0, 18:1 PC/PG) lipid bilayers reveal a preference for antiparallel double helix (ADH) formation. The adoption of nascent conformers for both GA analogues was observed using MT-ESI for samples associated with DMPC/DMPG (12:0 PC/PG) bilayers.