The analysis of cell cycle in cancer cell lines provides valuable information for the diagnosis of cancer. Cell cycle synchronization is an effective way to analyze different cell cycle phases. In this study, both asynchronous and synchronous ER (+) MCF-7 and ER (-) MDA-MB-453 breast cancer cells were analyzed. Cells were stained using PI and asynchronous cell cycle phases were analyzed by flow cytometer. Then both cell types were synchronized close to G1-S phase using 2 mM Hydroxyurea and synchronous cell cycle phases were analyzed. The asynchronous cells moved smoothly and completed their cycle in 24 h. The significant inhibition of the DNA synthesis in synchronous MCF-7 cells was observed at 12 h after the removal of Hydroxyurea, and almost 28 % of the cells were at S phase whereas only 58 % of the cells were in G1 phase. A marked G1/S accumulation also was observed in synchronous MDA-MB-453 cells immediately after removal of Hydroxyurea at 0 h. The DNA synthesis was significantly blocked because % 82 of the cells was at G1 phase whereas only % 10 of the cells was at S phase. DNA synthesis resumed and cells returned to normal cell cycle over the next 48 h. There was no significant “sub G1 peak” indicating apoptosis or cell death. This data indicates that Hydroxyurea may be used to synchronize both cells without inducing apoptosis or cell death