HeLa is one of the oldest and most commonly used cell lines in biomedical research. Owing to the ease of which they can be effectively synchronized by various methods, HeLa cells have been used extensively for studies of the cell cycle. Here we describe several protocols for synchronization of HeLa cells from different phases of the cell cycle. Synchronization in G(1) phase can be achieved with the HMG-CoA reductase inhibitor lovastatin, S phase with a double thymidine block procedure, and G(2) phase with the CDK inhibitor RO3306. Cells can also be enriched in mitosis by treating with nocodazole and mechanical shake-off. Release of the cells from these blocks enables researchers to follow gene expression and other events through the cell cycle. We also describe several protocols, including flow cytometry, BrdU labeling, immunoblotting, and time-lapse microscopy, for validating the synchrony of the cells and monitoring the progression of the cell cycle after release.