Simple SummaryNeuroblastoma is a common childhood cancer with poor prognosis. Prior studies suggest that inhibition of molecules called checkpoint proteins, which normally prevent one’s own immune system from attacking itself, has been successfully used for treatment of multiple advanced adult cancers but has yet to be fully explored in neuroblastoma. Cancer can hijack these pathways to prevent the immune system from recognizing and destroying cancer cells. We investigated checkpoint protein expression in pediatric neuroblastoma and its role in drug resistance. We created drug-resistant neuroblastoma cell lines and compared expression of checkpoint proteins between drug-resistant and parental cell lines. In total, 13 checkpoint proteins were expressed by all cell lines regardless of drug resistance. Although PD-L1 and checkpoint proteins do not necessarily impart drug resistance, they may be potential targets for drug therapy. Benchmarking checkpoint proteins provides the basis for future studies identifying targets for directed therapy and biomarkers for cancer detection or prognosis.Neuroblastoma is a common childhood cancer with poor prognosis when at its advanced stage. Checkpoint molecule inhibition is successful in treating multiple advanced adult cancers. We investigated PD-L1 and other checkpoint molecule expression to determine their roles in drug resistance and usefulness as targets for drug therapy. We developed three doxorubicin-resistant (DoxR) cell lines from parental cell lines. Matrigel in vitro invasion assays were used to compare invasiveness. Western blot assays were used to compare PD-L1 expression. Immuno-oncology checkpoint protein panels were used to compare concentrations of 17 checkpoint molecules both cellular and soluble. PD-L1 and 12 other checkpoint molecules were present in all cell lysates of each cell line without significantly different levels. Three were solubilized in the media of each cell line. PD-L1 is expressed in all DoxR and parental neuroblastoma cells and may be a potential target for drug therapy although its role in drug resistance remains unclear. Benchmarking checkpoint molecules provides the basis for future studies identifying targets for directed therapy and biomarkers for cancer detection or prognosis.