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%. Synthetic LNPs are commonly composed of four components; an ionizable lipid, a helper lipid, cholesterol and a poly(ethylene glycol)ylated (PEG) lipid. Ionisable lipids interact electrostatically with the negatively charged mRNA facilitating its encapsulation within the LNP. These also enable endosomal escape by appropriate pKa choice. Helper lipids, such as the phospholipids distearoylphosphatidylcholine (DSPC) and dioleoylphosphatidylethanolamine (DOPE), contribute to the structural integrity and stability of LNPs. Cholesterol is incorporated into LNPs to modulate their fluidity and rigidity, enhancing particle stability and membrane fusion capabilities and also helps maintain the structural integrity of LNPs, allowing for delivery and release of mRNA into target cells. The inclusion of PEG-lipids on the LNP surface contributes to both formulation stability, and the 'stealth' properties of the nanoparticles, with longer fatty acids allowing them to circulate in the bloodstream longer and increasing the chances of reaching their target cells whilst shorter fatty acids are shed faster enabling the lipid surface to be exposed and absorb the serum proteins. The study of the structural, compositional, and functional aspects of LNPs for drug delivery has employed various analytical and imaging techniques. Cryo-EM allows for the observation of the nanoparticle morphology and internal structure, providing insights into the lipid organization, but without information on molecular identity. Small-angle neutron scattering (SANS) combined with deuteration of components at large scale facilities has been used to quantify the lipid distribution at the surface of LNPs using isotopic contrast variation. Small-angle X-ray scattering (SAXS) using synchrotron sources, has been used to probe bulk phase structures. Dynamic light scattering (DLS) is used to rapidly measure the size distribution and polydispersity index (PDI) of LNPs, offering insights into their homogeneity and stability. NMR spectroscopy provides information on the molecular structure, dynamics, and interactions within LNPs. It can be used to study the composition of the lipid core, the conformation of PEG chains, and the interaction between PEG and other components of LNPs, however the structural analysis of LNPs is limited by NMR sensitivity. To provide a method that can address these uncertainties, here we show that molecular depth profiling of LNPs is possible using cryogenic OrbiTrap 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 within the vacuum of the Cryo-OrbiSIMS instrument, verified by analysis using cryo-transmission electron microscopy (Cryo-TEM) on the same sample.