•A validated method for selectively quantitating α-GalCer in DPPC/Chol liposomes was developed.•We have demonstrated the application of the method for determining incorporation efficiency with varying α-GalCer concentrations.•The method has utility for monitoring the stability of α-GalCer incorporation in liposome formulations.α-Galactosylceramide (α-GalCer) is a synthetic glycosphingolipid that has been widely studied as a potential vaccine adjuvant and exhibits distinct immunoregulatory effects when incorporated into liposomes. The aim of this study was to utilise the high selectivity of liquid chromatography–tandem mass spectrometry (LC–MS/MS) to develop a validated method for quantitating α-GalCer incorporation into liposomes.To separate unincorporated α-GalCer, liposomes were pelleted by centrifugation, resuspended and mixed with the mobile phase to allow solubilisation of the liposome components. Separation of α-GalCer was achieved using a C8 column with a mobile phase consisting of 7:3 (v/v) ethanol:methanol with 0.5% formic acid and 10 mM ammonium formate. The multiple reaction monitoring (MRM) mode for the triple quadrupole was used to measure the peak area of the m/z 858.74 → 696.69 transition, which is the change in mass of the α-GalCer molecule after detachment of the galactose group through fractionation. The method developed here demonstrated specificity for α-GalCer in the presence of other liposome components.The validated method was used to determine the incorporation of α-GalCer in dipalmitoylphosphatidylcholine (DPPC) liposomes at varying concentrations and to monitor the stability of liposomes containing α-GalCer during storage at 4 °C during 30 days.