Here we describe several methods for the characterization of epithelial–mesenchymal transition (EMT) at the cellular, molecular and behavioral level. This protocol describes both in vitro and in vivo approaches designed to analyze different features that when taken together permit the characterization of cells undergoing transient or stable EMT. We define straightforward methods for phenotypical, cellular and transcriptional characterization of EMT in vitro in monolayer cultures. The procedure also presents technical details for the generation of in vitro three-dimensional (3D) cultures analyzing cell phenotype and behavior during the EMT process. In addition, we describe xenotransplantation techniques to graft 3D cell cultures into mice to study in vivo invasion in a physiological-like environment. Finally, the protocol describes the analysis of selected EMT markers from experimental and human tumor samples. This series of methods can be applied to the study of EMT under various experimental and biological situations. Once the methodology is established, the time required to complete the protocol may vary from 3 to 4 weeks (monolayer cultures) and up to 6–8 weeks if including 3D cultures.