Chemotherapy of cancer experiences a number of shortcomings including development of drug resistance. This fact also holds true for neuroblastoma utilizing chemotherapeutics as vincristine. We performed a comparative analysis of molecular and cellular mechanisms associated with vincristine resistance utilizing cell line as well as human tissue data. Differential gene expression analysis revealed molecular features, processes and pathways afflicted with drug resistance mechanisms in general, and specifically with vincristine significantly involving actin associated features. However, specific mode of resistance as well as underlying genotype of parental, vincristine sensitive cells apparently exhibited significant heterogeneity. No consensus profile for vincristine resistance could be derived, but resistance-associated changes on the level of individual neuroblastoma cell lines as well as individual patient profiles became clearly evident. Based on these prerequisites we utilized the concept of synthetic lethality aimed at identifying hub proteins which when inhibited promise to induce cell death due to a synthetic lethal interaction with down-regulated, chemoresistance associated features. Our screening procedure identified synthetic lethal hub proteins afflicted with actin associated processes holding synthetic lethal interactions to down-regulated features individually found in all chemoresistant cell lines tested, therefore promising an improved therapeutic window. Verification of such synthetic lethal hub candidates in human neuroblastoma tissue expression profiles indicated the feasibility of this screening approach for addressing vincristine resistance in neuroblastoma.