Confocal and two-photon microscopy has been widely used in bone research to not only produce high quality, three-dimensional images but also to provide valuable structural and quantitative information. In this article, we describe step-by-step protocols for confocal and two-photon microscopy to investigate earlier cellular events during colonisation of cancer cells in bone using xenograft mouse models. This includes confocal/two-photon microscopy imaging of paraformaldehyde fixed thick bone sections and frozen bone samples.