Neuroblastoma, the commonest solid extracranial tumour of infancy, is treated with a number of therapeutic modalities. Depending upon staging at presentation, these include resection, “differentiation therapy” with retinoid and related morphogens and conventional chemotherapy. Latterly targeted immunotherapy has been directed at a defined ganglioside GD2 specifically enriched in the plasma membrane (PM) of aberrant cells. This targeted monoclonal antibody approach, while offering promise clinically is compromised by expression of the relevant ganglioside on nerve cells among others which also bind inducing significant pain. The ameliorative opiate therapy administered is both undesirable and potentially harmful. Clearly, a more restricted target would offer the possibility of minimising this undesirable side-effect and obviate the need for morphia. Recent advances in glycolipidomics now permit the extension ESI-MS-based, small molecule analyses to these molecules with unparalleled sensitivity and discrimination. Application of these methodologies to cultured neuroblastoma cells should enable a more accurate identification of the composition of neuroblastoma PM gangliosides with the aim of tailoring the immunotherapeutic approach more rigorously in future. Here we have used analytical and imaging approaches to define the pattern and topology of ganglioside expression at the PM. Analytically we have used Qtof mass spectrometry to characterise fragmentation patterns of major ganglioside species from standards and tissue extracts. This has allowed us to identify the diverse range of ganglioside species even those with relatively low abundance from their unique profiles and will enable us to confirm the identities of the “unusual” once we have completed analyses of control neural tissues. For imaging work we used a panel of recently commercially available monoclonal antibodies to 13 gangliosides in immunofluorscence studies of cultured Kelly and SK-N-AS human neuroblastoma cell lines. This approach should provide the complementary data that helps us to identify which gangliosides are displayed on the outer aspect of the PM in contrast to what is present within the whole cell. Together these data may provide evidence to accurately define more viable candidate ganglioside molecular targets or combinations of ganglioside targets for neuroblastoma therapy.