While standards for depth profile resolution estimation for LNP components are not readily available, supported lipid bi-layers could be envisaged which would have the advantage of being flat, or multi-component micelles, which would well represent the particulate geometry and approximate dimensions. This would allow the variability in PEG overlayer thickness inferred from the broadness of profiles seen herein to be ascribed to actual or experimental depth resolution limits. The new analytical insights this novel method can achieve can be paired with endosomal mimics to examine the influence on LNP components in endosomal escape, offering a powerful framework for to improve therapeutic formulations. In summary, we find that Cryo-OrbiSIMS depth profiling offers a valuable methodology for label free characterisation of the native LNP surface molecular structure and stratification in the frozen hydrated state. In addition to this high-fidelity insight into the surface molecular stratification of the individual components, the orientation of the PEG-lipid can be identified, consistent with models form neutron scattering and well-established models of similar LNPs.