Most breast cancer originates in the lobules or ducts of the breast. It is the second leading cause of death and the most common type of cancer in women worldwide. Extensive studies have been conducted to treat breast cancer, and various diseases and parameters related to this disease have been intensively evaluated in the studies of the last decade. Quercetin is a high amount of flavonoids found in plants, especially vegetables and fruits. It is found in different amounts in many foods, especially cruciferous vegetables, grapes, apples, tomatoes and blueberries. Quercetin shows promise as a potential anticancer agent. This study aimed at determining the antiproliferative and apoptotic effects of the flavonoid called Quercetin, which is known to have anticancer properties, on breast cancer cell lines in monolayer (2D) and multicellular spheroid (3D) culture. TUNEL, BrdU, Endo-G, and Caspase-3 immunohistochemical studies in both 2D and 3D culture systems were used to examine the quercetin effects on carcinoma cell lines of human breast MCF-7 and MDA-MB-231. Also, to determine some gene expression changes (Endo-G, Caspase-3, Caspase-8, and Caspase-9) participating in apoptosis, a qRT-PCR study was conducted. In both 2D and 3D culture conditions, Quercetin induces apoptosis and suppresses cell proliferation. In addition, these results are compatible with gene expression results. In cancer treatment, Quercetin can be considered a potential anticancer agent.