MYCN gene amplification clearly correlates with poor prognosis in patients with neuroblastoma and some types of cancer. Basically transcriptional factors are thought to be “un-druggable” targets, and therefore, alternative approaches are required to develop new therapeutics for this disease. Synthetic lethal (SL) approaches are emerging as a promising strategy for cancer therapy. For instance, poly ADP-ribose polymerase (PARP) inhibitor “Olaparib” has been approved for treating BRCA-deficient ovarian cancer in EU and USA. The double-stranded DNA breaks arising from the inhibition of PARP cannot be repaired in BRCA-deficient cells, which leads to the cancer cell-specific cell death. This approach is thought to be effective in MYCN-amplified neuroblastoma cells with elevated endogenous DNA damage caused by dysregulated gene expression. In order to identify SL genes in MYCN-amplified neuroblastoma cells, we performed a genome-wide shRNA library screening. The commercial library that consists of over 80,000 shRNA constructs targeting about 16,000 human genes was used. IMR-32 cells (MYCN-amplified) and SH-SY5Y cells (MYCN single copy) were transduced with lentiviruses carrying the shRNA sequences. The relative abundance of shRNA constructs in each cell line is then quantified by next-generation sequencing (MiSeq; Illumina Inc.). shRNA sequences with a false discovery rate <0.1 from an exact test analysis in edgeR software were selected. In addition to already known synthetic lethal genes (e.g. SMC2, CSNK1E), about 130 genes were identified as new candidates. Based on our experimental validations using siRNA or chemical inhibitors, some mitotic kinases were proposed to be new SL genes in MYCN-amplified neuroblastoma cells. The molecular mechanisms underlying the lethality and its potential as a drug target will be discussed.