Billions of doses of mRNA-loaded lipid nanoparticle (LNP) vaccines represent a spectacular recent example of the utility of therapeutic nanomaterials. The molecules presented on the surface of such LNPs influence how the material interacts with the environment inside the body, including which biomolecules it adsorbs to form the biomolecular corona. This biointerface is critical in determining nanoparticle trafficking, potency and immunological reaction, yet uncertainties surrounding LNP structural organisation, such as the location of ionisable lipids either in the core or in the exterior shell, remain. Such structural details likely have implications on nanoparticle behaviour, including the critical stage of endosomal escape in transfection, for which efficiencies are estimated to be as low as 2%. To provide a method that can address these uncertainties, we here show that molecular depth profiling of LNPs is possible using cryogenic OrbiTrap TM secondary ion mass spectrometry (Cryo-OrbiSIMS). We find this laboratory-based direct analysis method can identity the molecular structure of the LNP surface, revealing the relative depths of lipid and RNA components. Furthermore, the orientation of certain molecules can even be discerned where fragments resulting from different portions of the molecule are detected. This information relies on retention of the native hydrated structure in ice within the vacuum of the Cryo-OrbiSIMS instrument, verified by analysis using transmission electron microscope (TEM) on the same sample.