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Simple Summary: Breast cancer is the type of cancer that most affects women worldwide, and until today, it is difficult to find an effective treatment against this disease. Scientists are exploring new ways to test treatments using systems in the laboratory capable of mimicking tumors, called 3D models or spheroids. This study aimed to understand how breast cancer spheroids behave and respond to a common drug used in the clinic to treat cancer, doxorubicin. Understanding these processes could lead to improved treatments for breast cancer and other types of cancer. We found that the spheroids showed close features of real tumors and showed changes in proteins associated with cancer spread (metastasis). When the spheroids were treated with doxorubicin, the size of the spheroids was reduced, cells died, and the spread of breast cancer cells was also reduced. These results suggest, for the first time, that doxorubicin could be a good candidate to help stop cancer metastasis, which can be further studied. Breast cancer is the most prevalent cancer among women worldwide. Therapeutic strategies to control tumors and metastasis are still challenging. Three-dimensional (3D) spheroid-type systems more accurately replicate the features of tumors in vivo, working as a better platform for performing therapeutic response analysis. This work aimed to characterize the epithelial–mesenchymal transition and doxorubicin (dox) response in a mammary tumor spheroid (MTS) model. We evaluated the doxorubicin treatment effect on MCF-7 spheroid diameter, cell viability, death, migration and proteins involved in the epithelial–mesenchymal transition (EMT) process. Spheroids were also produced from tumors formed from 4T1 and 67NR cell lines. MTSs mimicked avascular tumor characteristics, exhibited adherens junction proteins and independently produced their own extracellular matrix. Our spheroid model supports the 3D culturing of cells isolated from mice mammary tumors. Through the migration assay, we verified a reduction in E-cadherin expression and an increase in vimentin expression as the cells became more distant from spheroids. Dox promoted cytotoxicity in MTSs and inhibited cell migration and the EMT process. These results suggest, for the first time, that this model reproduces aspects of the EMT process and describes the potential of dox in inhibiting the metastatic process, which can be further explored. [ABSTRACT FROM AUTHOR]