Three-dimensional (3D) tumour spheroids are utilized in cancer research as a more accurate model of the in vivo tumour microenvironment, compared to traditional two-dimensional (2D) cell culture. The spheroid model is able to mimic the effects of cell-cell interaction, hypoxia and nutrient deprivation, and drug penetration. One characteristic of this model is the development of a necrotic core, surrounded by a ring of G1 arrested cells, with proliferating cells on the outer layers of the spheroid. Of interest in the cancer field is how different regions of the spheroid respond to drug therapies as well as genetic or environmental manipulation. We describe here the use of the fluorescence ubiquitination cell cycle indicator (FUCCI) system along with cytometry and image analysis using commercial software to characterize the cell cycle status of cells with respect to their position inside melanoma spheroids. These novel methods may be used to track changes in cell cycle status, gene/protein expression, cell viability, cell morphology or even cell migration in different sub-regions of tumour spheroids over time and under different conditions. We have also tested the benefits and drawbacks of different methods of imaging 3D spheroids including confocal, multi-photon and light-sheet microscopy.