Cells cultured in 2D can differ in terms of both physiology and cellular responses compared with cells in vivo. This has led to a surge in the popularity of using 3D culture techniques as mounting evidence suggests that culturing cells in 3D is more representative of the in vivo environment, even to the extent that the gene expression profiles of cells from 3D cultures more accurately reflect clinical expression profiles than those observed in 2D cultures. 3D culture offers the potential for more accurate models of drug delivery and efficacy, as well as numerous clinical and research applications, and is becoming increasingly capable of integrating into high-throughput activities. Spheroids, or sphere cultures, have become an especially exciting area of 3D in vitro culture due to their great potential for use in studies that investigate growth and function of both malignant and normal tissues. These sphere cultures have contributed considerably to our knowledge of cellular responses thanks to the accuracy with which they reflect the in vivo system.