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Quantification of the lipid content in liposomal adjuvants for subunit vaccine formulation is of extreme importance since this concentration impacts both on efficacy and stability. Within this paper we outline an HPLC-ELSD method that allows for the rapid and simultaneous quantification of lipid concentrations within liposomal systems prepared by three liposomal manufacturing techniques (lipid film hydration, high shear mixing and microfluidics). The ELSD system was used to quantify 4 lipids: 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), cholesterol, dimethyldioctadecylammonium (DDA) and D-(+)-trehalose 6,6’-dibehenate (TDB). The developed method offers rapid, high sensitivity, direct linearity and a good consistency on the responses (R2>0.993 for 4 lipids tested). The corresponding LOD and LOQ were 0.11 and 0.36 mg/mL (DMPC), 0.02 and 0.80 mg/mL (cholesterol), 0.06 and 0.20 mg/mL (DDA), and 0.05 and 0.16 mg/mL (TDB), respectively. HPLC-ELSD was shown to be a rapid and effective method for the quantification of lipids within liposome formulations without the need for lipid extraction processes.