17β-estradiol (E2), the main circulating estrogen hormone, is a critical signaling factor for the growth, differentiation, and function of breast epithelial cells. The effects of E2 on the breast tissue are primarily mediated by the estrogen receptor α (ERα). Deregulation of the E2-ERα signaling contributes to the initiation/progression of breast cancer and resistance to treatments. Cell lines from breast adenocarcinomas as in vitro model systems provide invaluable insight into cellular events, drug discovery, and drug resistance. Among ERα-synthesizing cell lines, MCF7 and T47D cells are widely used to elucidate cell cycle phase-specific molecular events that coordinate cellular proliferation mediated by E2-ERα. Due to variable results in generating phase-enriched populations with various approaches, we wanted to reassess cell cycle synchronization-coupled phase enrichment with charcoal dextran-treated fetal bovine serum, CD-FBS, as an effective hormone withdrawal approach, alone or in combination with excess thymidine, as a DNA replication inhibitor, and/or nocodazole, a microtubule poison, in MCF7 and T47D cells. We find that hormone withdrawal synchronizes both MCF7 and T47D cells at the G0/G1 phase. Supplementation of CD-FBS with E2 enriches the S phase population. E2 with nocodazole and nocodazole-coupled mitotic shake-off augments the G2/M phase population of MCF7 cells. However, the double thymidine block approach with nocodazole or nocodazole-coupled mitotic shake-off is more effective in enriching S and G2/M phase populations of T47D cells. Our results highlight the differential efficacy of synchronization approaches in MCF7 and T47D cells that could provide a framework for cell cycle-specific applications in breast cancer research.