The intrinsic heterogeneity of many nanoformulations is currently challenging to characterise on both the single particle and population level. Therefore there is great opportunity to develop new techniques to describe and understand nanomedicine heterogeneity, which will aid translation to the clinic by informing manufacturing quality control, characterisation for regulatory bodies, and connecting nanoformulation properties to clinical outcomes to enable rational design. Here, we present an analytical technique to provide such information, whilst measuring the nanocarrier and cargo simultaneously with label-free, non-destructive single particle automated Raman trapping analysis (SPARTA). We first synthesised a library of model compounds covering a range of hydrophilicities and providing distinct Raman signals. These compounds were then loaded into model nanovesicles (polymersomes) that can load both hydrophobic and hydrophilic cargo into the membrane or core regions respectively. Using our analytical framework, we characterised the heterogeneity of the population by correlating the per particle membrane and cargo signals. We found that core and membrane loading can be distinguished, and we detected sub-populations of highly loaded particlesin certain cases. We then confirmed suitability of our technique in liposomes, another Peer reviewed version of the manuscript published in final form in ACS Nano (2023) 2 nanovesicle class, including the commercial formulation Doxil. Our label-free analytical technique precisely determines cargo location alongside loading and release heterogeneity in nanomedicines, which could be instrumental for future quality control, regulatory body protocols and development of structure-function relationships, to bring more nanomedicines to the clinic