Melanoma is a malignant skin cancer developed from melanocytes. Treatment resistance and disease recurrence have been linked to the survival of cancer stem cells (CSCs). CSCs are highly proliferative, self-renewing cells able to differentiate into heterogeneous populations that maintain some similarities with their progenitors. This study aimed to develop three-dimensional melanosphere models containing CSCs cells that serve as a better mimic of solid tumors than other cell culture models. Melanospheres were prepared with 100 or 1000 cells/well, under non-adherent conditions using 30 µL of 2% agarose per well. Melanosphere morphology was analyzed by scanning electron microscopy (SEM) and light microscopy. For both conditions, a significant increase in melanosphere volume was observed on the 7^th day compared with the 4^th day of culture. Melanospheres also exhibited high cell viability and ability to form new colonies. The presence of CD271-, CD133-, and CD44-positive cells was analyzed by flow cytometry. Melanospheres showed a higher percentage of CSC markers than 2D cell cultures. Also, melanospheres cultured with lower densities showed major presence of CD44+, while higher density melanospheres showed major presence of CD271+. The findings suggest that different densities of cells are capable of enriching distinct markers of CSC in melanospheres, as an effective strategy to facilitate cancer research and therapy development.