Quantification of the lipid content in liposomal adjuvants for subunit vaccine formulation is of extreme importance, since this concentration impacts both efficacy and stability. In this paper, we outline a high performance liquid chromatography-evaporative light scattering detector (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 four lipids: 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), cholesterol, dimethyldioctadecylammonium (DDA) bromide, and ᴅ-(+)-trehalose 6,6′-dibehenate (TDB). The developed method offers rapidity, high sensitivity, direct linearity, and a good consistency on the responses (R2 > 0.993 for the four lipids tested). The corresponding limit of detection (LOD) and limit of quantification (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.