There is growing interest in using three-dimensional (3D) cell structures to model tumors, organs, and tissues to accelerate translational research. Significant advances have been made in creating such structures to recreate the in vivo environment and perform complex assays against them for a better understanding of the human disease. More recently, automation has been employed to reduce manual intervention, increase throughput, and improve assay precision.  In this Methods Collection, we will focus on three critical areas of technology development related to 3D cell-based assays.  First, is the generation of 3D cell models such as spheroids, organoids, and micro-tissues. Topics include techniques and automation methods to improve the efficiency, uniformity, and throughput of 3D cell model formation.  The second is novel assay methods and instrumentation to improve assay performance.  Included are ways to handle and manipulate 3D cell models, microfluidic-based systems, and techniques to improve assay precision and reproducibility.  The last area is the optimization of sample interrogation and data acquisition methods. This topic includes methods for measurement of cell viability in 3D structures, characterization of metabolism dynamics, and 3D high content imaging and analysis.  This collection will showcase the latest methods for 3D cell-based assays being used for research in a wide array of therapeutic areas such as oncology, neuroscience, metabolic diseases, and gastrointestinal disease.  These methods are also becoming more commonly employed in drug discovery and development, especially in compound screening and ADME/Tox profiling.