Two-dimensional (2D) cell culture is the prime methodology used, for screening anticancer therapeutics. However, when 2D cellular models are used, the architecture of native tumors is not fully represented, leading in some cases to an unsuccessful prediction of cancer cells response to drugs. On the other hand, there is a need to reduce the use of animal research models once they have economical and ethical problems associated. To overcome the limitations associated to 2D cell culture models and in vivo assays, the researchers started to perform cell growth in three-dimensions (3D), for reproducing in vitro the 3D structure of solid tumors. One of the most applied techniques to produce these 3D cellular aggregates, also known as spheroids, is Liquid Overlay Technique (LOT), in which cells are forced to aggregate due to their limited adhesion to certain biomaterials, usually agarose or agar. However, these biopolymers cannot interact with cancer cells, neither establish interactions that are similar to those occurring between cells and extracellular matrix (ECM) in solid tumors, which are responsible for the activation of cellular signaling pathways that regulate cancer cells behavior. In order to mimic not only the 3D structure but also the cell-ECM interactions that occur in tumors, it has been proposed the production of 3D models in which cells can interact with tumor ECM components. One of the biomaterial that has been used with this objective is the hyaluronic acid (HA). This compound is one of the main constituents of tumor ECM, it avoids cell adhesion and it has an essential role in cancer progression. In this work it was optimized, for the first time, the coating of surfaces with HA that were used for the production of reproducible heterotypic breast cancer spheroids. The obtained results revealed that the HA coated surfaces allow the production of spheroids that reproduce the 3D structure and the cellular heterogeneity presented by breast solid tumors. Furthermore, it was possible to control the size, shape and number of spheroids produced by changing the HA concentration and the number of cells initially seeded. Overall, these breast cancer spheroids assembled on HA-coated surfaces represent a huge improvement for the future development of anticancer therapies.