Microfluidics is a powerful technology that allows the production of cationic liposomes by the hydrodynamic focusing method. We first studied a single hydrodynamic focusing (SHF) device, which uses a central stream in which lipids dispersed in ethanol are injected and hydrodynamically compressed by the two aqueous streams. The ethanol diffusion from the inner stream to the aqueous stream encourages the formation of the liposomes. To intensify the mass diffusion and increase the surface area between the two fluids, a second device was designed with double hydrodynamic focusing (DHF). We investigated the influence of fluid flow velocity (V-f), Flow Rate Ratio (FRR) and total lipid concentration (C-lip) on the particle size of the CLs produced. The DHF microfluidic device had the ability of using higher V-f values than the SHF device, which resulted in a higher productivity level. Small Angle X-ray Scattering (SAXS) experiments were performed to structurally characterize the cationic liposomes produced by both microfluidic devices. The SAXS results revealed that both devices produce unilamellar cationic liposomes with a very small fraction of multilamellar liposomes; this finding is in agreement with the observations made in the analysis of the liposomes using Transmission Electron Microscopy (TEM). The biological efficacies of the cationic liposomes produced by both microfluidic devices were examined in vitro in HeLa cells, which confirmed their potential for gene delivery and vaccine therapy applications. (C) 2013 Elsevier B.V. All rights reserved.