Neuroblastoma is a solid, intractable, extracranial, pediatric tumor with complex, heterogeneous clinical behaviors. Chemotherapy is widely used to treat neuroblastoma and many studies have attempted to decipher the action mechanism of anticancer drugs; however, the dose-dependent responses of neuroblastoma cells to anticancer drugs in various microenvironments have not been fully explored. Here, we investigated the effects of different topotecan concentrations on human neuroblastoma SK-N-SH cells under varying nutrient supply conditions. The survival rate of serum-starved SK-N-SH cells increased upon treatment with a high concentration (1 μM) of topotecan. Using quantitative proteomics and network analysis, we showed that topotecan (1 μM) upregulated DNA repair and cholesterol-mediated topotecan efflux, resulting in topotecan resistance. Time-resolved topotecan quantitation using multiple reaction monitoring mass spectrometry further supports that topotecan resistance was associated with enhanced drug efflux. Our results provide a model for high-dose-dependent chemoresistance in neuroblastoma cells that could enable a patient-dependent chemotherapy screening strategy.