Established cancer cell lines have and continue to contribute tremendously for many discoveries and are some of the important tools for studies involving drug and biomarker discovery. Cell culture techniques has seen a multifold change with advancements in the technology; the biocompatible material available as culture supports and with enhancing understanding of cell physiology and culture behaviors. Culturing cells as monolayers in the 2D system are extremely important for various applications, likewise, the 3D systems of cell cultures have thrown valuable insights which were not possible with the 2D systems. Culturing cells in a 3D model is considered to bring in a closer ‘in vivo’ effect to the cells and cells in such a system sure show marked changes compared to their 3D counterparts. Several matrices and supports are available for culturing cells in 3D. Agarose hydrogels offer us a useful combination of economy and ease of applications. We present here the uses of agarose hydrogels to obtain 3D aggregates of 3 different human cancer cell lines as three chapters and one chapter dedicated to the model applications of such a system.