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Summary: We describe a three-dimensional (3D) in vitro assay for quantifying cancer cell invasion into a 3D microenvironment with defined biochemical and biophysical properties. Researchers can quantify invasion dynamics (e.g., cell motility and directionality) and examine morphological changes during invasion, using live-cell and confocal imaging techniques. Together, these advantages over existing in vitro invasion assays, such as transwell-based assays, provide researchers with a valuable tool to gain insight into the mechanisms regulating cancer cell invasion.For complete details on the use and execution of this protocol, please refer to Padilla-Rodriguez et al. (2018) and Watson et al. (2021). : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.