Recurrent and metastatic cancer lesions often undergo a period of dormancy, which is closely associated with cancer cell quiescence, a state whereby cancer cells exit the cell cycle and are reversibly arrested in G0 phase. Quiescent cancer cells are inherently resistant to cell death and refractory to therapeutic drugs. However, the mechanisms responsible for the resistance remain largely undefined. This is closely associated with the lack of understanding of their biological properties as a consequence of technical hurdles in the isolation and analysis of viable quiescent cells. Nevertheless, it is known that quiescent cells are characteristically negative for the proliferation marker Ki67 and express high levels of the cyclin-dependent kinase (CDK) inhibitor p27. In this study, we developed a CRISPR/Cas9-based system to fuse a green fluorescent protein (EGFP) gene with endogenous CDKN1B, the gene encoding p27, and a red fluorescent protein (mCherry) gene with endogenous MKI67, the gene encoding Ki67 in the genome of human melanoma cells. By using this system, we have successfully isolated viable p27high/Ki67- (quiescent) melanoma cells using fluorescence-activated cell sorting (FACS). The quiescent state of these cells was confirmed by dual nucleic acid staining (DNA with Hoechst -33342, and RNA with Pyronin Y). The isolated quiescent cells and cycling cells were subjected to comparative RNA-seq and proteomics analyses. These omics results have revealed molecular networks involved in regulating cell quiescence, which are promising hints and can be confirmed as targets to overcoming resistance of quiescent melanoma cells to treatment. In addition, the established cells line can be used together with screening libraries to select the drugs which specifically target quiescent cancer cells.