Subunit vaccines require particulate adjuvants to induce the desired immune responses. Pre-clinical manufacturing methods of adjuvants are often batch dependent, which complicates scale-up for large-scale good manufacturing practice (GMP) production. The cationic liposomal adjuvant CAF09b, composed of dioctadecyldimethylammonium bromide (DDA), monomycoloyl glycerol analogue 1 (MMG) and polyinosinic:polycytidylic acid [poly(I:C)], is currently being clinically evaluated in therapeutic cancer vaccines. Microfluidics is a promising new method for large-scale manufacturing of particle-based medicals, which is scalable from laboratory to GMP production, and a protocol for production of CAF09b by this method was therefore validated. The influence of the manufacture parameters [Ethanol] (20–40% v/v), [Lipid] (DDA and MMG, 6–12 mg/mL) and dimethyl sulfoxide [DMSO] (0–10% v/v) on the resulting particle size, colloidal stability and adsorption of poly(I:C) was evaluated in a design-of-experiments study. [Ethanol] and [DMSO] affected the resulting particle sizes, while [Lipid] and [DMSO] affected the colloidal stability. In all samples, poly(I:C) was encapsulated within the liposomes. At [Ethanol] 30% v/v, most formulations were stable at 21 days of manufacture with particle sizes <100 nm. An in vivo comparison in mice of the immunogenicity to the cervical cancer peptide antigen HPV-16 E7 adjuvanted with CAF09b prepared by lipid film rehydration or microfluidics showed no difference between the formulations, indicating adjuvant activity is intact. Thus, it is possible to prepare suitable formulations of CAF09b by microfluidics.