〔Abstract〕 Objective To establish a method for detecting the duration of the synthesis-phase(Ts) and the total cell-cycle(Tc) based on the dual labeling of 5-ethynyl-2′deoxyuridine(EdU) and 5-Bromodeoxyuridinc(BrdU). Methods HEK293T cells were double labeled with EdU and BrdU and then subjected to immunofluorescence staining. The fluorescence signals were collected using a High-Content Imaging System. The numbers of EdU-positive and BrdU-positive cells in the entire cell population were counted. Based on the cell cycle calculation formula in a previously published article, it was improved to accurately calculate the duration of Ts and Tc of cells. The cell cycle time of human embryonic stem cells was calculated using the established method. Results Based on the kinetic parameters of the EdU/BrdU dual labeling, the Ts of human embryonic stem cells was 10.9 h, and the Tc was 15.3 h. Conclusion The EdU/BrdU dual labeling method can be used to detect the duration of the cell cycle, providing an accurate and efficient method for cell cycle analysis.