It is of crucial importance to determine the concentration of the different components in the formulation accurately, during production. In this respect, near-infrared (NIR) spectroscopy represents an intriguing alternative that offers rapid, non-invasive and non-destructive sample analysis. This method, combined with multivariate data analysis was successfully applied to quantify the total concentration of lipids in the liposomal CAF01 adjuvant, composed of the cationic surfactant dimethyldioctadecylammonium bromide (DDA) and the immunomodulator alpha,alpha'-trehalose 6,6'-dibehenate (TDB). The near-infrared (NIR) detection method was compared to a validated high-performance liquid chromatography (HPLC) method and a differential scanning calorimetry (DSC) analysis, and a blinded study with three different sample concentrations was performed, showing that there was no significant difference in the accuracy of the three methods. However, the NIR and DSC methods were more precise than the HPLC method. Also, with the NIR method it was possible to differentiate between various concentrations of trehalose added as cryo-/lyoprotector. These studies therefore suggest that NIR can be used for real-time process control analysis in the production of CAF01 liposomes.