Cell cycle analysis is important for cancer research. We present herein a novel method for accurate cell cycle analysis. This method analyzes the cell cycle by multiparameter flow cytometry based on simultaneously labeling the cell nuclear DNA, RNA, and phosphorylated mitotic nuclei protein, using Hoechst 33342, pyronin Y, and MPM-2-Cy5, respectively, and our results demonstrated that this method could effectively divide the cell cycle into G0, G1, S, G2, and M phases. We further tested this method using the clinical anticancer agents crizotinib and taxol, and the results clearly illustrated that crizotinib and taxol arrested Jurkat cells in G0 and M phase, respectively. These results indicate that this method could be a very useful tool for cytokinetic and pharmacological research.