Traumas, fractures, and diseases can severely influence bone tissue. Insight into bone mineralization is essential for the development of therapies and new strategies to enhance bone regeneration. 3D cell culture systems, in particular cellular spheroids, have gained a lot of interest as they can recapitulate crucial aspects of the in vivo tissue microenvironment, such as the extensive cell-cell and cell-extracellular matrix (ECM) interactions found in tissue. The potential of combining spheroids and various classes of biomaterials opens also new opportunities for research within bone tissue engineering. Characterizing cellular organization, ECM structure, and ECM mineralization is a fundamental step for understanding the biological processes involved in bone tissue formation in a spheroid-based model system. Still, many experimental techniques used in this field of research are optimized for use with monolayer cell cultures. There is thus a need to develop new and improving existing experimental techniques, for applications in 3D cell culture systems. In this review, bone composition and spheroids properties are described. This is followed by an insight into the techniques that are currently used in bone spheroids research and how these can be used to study bone mineralization. We discuss the application of staining techniques used with optical and confocal fluorescence microscopy, molecular biology techniques, second harmonic imaging microscopy, Raman spectroscopy and microscopy, as well as electron microscopy-based techniques, to evaluate osteogenic differentiation, collagen production and mineral deposition. Challenges in the applications of these methods in bone regeneration and bone tissue engineering are described. STATEMENT OF SIGNIFICANCE: 3D cell cultures have gained a lot of interest in the last decades as a possible technique that can be used to recreate in vitro in vivo biological process. The importance of 3D environment during bone mineralization led scientists to use this cell culture to study this biological process, to obtain a better understanding of the events involved. New and improved techniques are also required for a proper analysis of this cell model and the process under investigation. This review summarizes the state of the art of the techniques used to study bone mineralization and how 3D cell cultures, in particular spheroids, are tested and analysed to obtain better resolved results related to this complex biological process.