Three-dimensional (3D) cell culture systems mimic the structural complexity of the tissue microenvironment that includes the extracellular matrix (ECM) in addition to the cellular components Thus, 3D culture systems are increasingly important as they resemble the ECM-cell and cell-cell physical interactions occurring in vivo. So far, several scaffold-based culture systems and techniques have been proposed as valuable approaches for large-scale production of spheroids, but often suffering of poor reproducible conditions or high costs of production. In this work we present a reliable 3D culture system based on collagen I-blended agarose hydrogels and show how the variation of the agarose weight percentage affects the physical and mechanical properties of the resulting hydrogel, being that with a lower amount of agarose more permeable, softer and more prone to degradation compared to hydrogels with higher agarose concentrations. We have also evaluated the effect of the different physical and mechanical properties of the agarose hydrogels on the growth, size, morphology and cell motility of spheroids obtained by culturing three different breast cancer cell lines (MCF-7, MDA-MB-361and MDA-MB-231). As proof of concept, the cisplatin penetration and its cytotoxic effect on the tumor spheroids was evaluated as function of the hydrogel stiffness. Noteworthy, the possibility to recover the spheroids from the hydrogels for further processing and other biological studies has been also considered.