Three-dimensional (3D) spheroids mimic important properties of tumors and may soon become a reasonable substitute for animal models and human tissue, eliminating numerous problems related to in vivo and ex vivo experiments and pre-clinical drug trials. Currently, various imaging methods including X-ray microtomography (micro-CT), exist but their spatial resolution is limited. Here, we visualized and provided a morphological analysis of spheroid cell cultures using micro-CT and compared it to that of confocal microscopy. An approach is proposed that can potentially open new diagnostic opportunities to determine the morphology of cancer cells cultured in 3D structures instead of using actual tumors. Spheroids were formed from human melanoma cell lines WM266-4 and WM115 seeded at different cell densities using the hanging drop method. Micro-CT analysis of spheroid showed that spheroid size and shape differed depending on the cell line, initial cell number, and duration of culture. The melanoma cell lines used in this study can successfully be cultured as 3D spheroids and used to substitute human and animal models in pre-clinical studies. The micro-CT allows for high-resolution visualization of the spheroids structure.Copyright © 2020. Published by Elsevier Ltd.