Changes in DNA replication during S‐phase can give insights into mechanisms of cell growth, cell cycle kinetics, and cytotoxicity. A common method for detection of cell proliferation utilizes the incorporation of a thymidine analog during DNA synthesis. Incorporation of multiple analogs at different time points can further define cell cycle kinetics. Traditionally, the dual‐pulse method has been done by combining 5‐bromo‐2′‐deoxyuridine (BrdU) with iododeoxyuridine or chlorodeoxyuridine, with detection using multiple cross‐reacting BrdU antibodies. This unit presents a dual‐pulse method using the thymidine analog 5‐ethyl‐2′‐deoxyuridine (EdU), detected by click chemistry, combined with BrdU labeling and detection. No cross reactivity with incorporated EdU is observed using the BrdU antibody clone MoBU‐1. EdU detection using click chemistry does not cross‐react with incorporated BrdU. Cells are first pulsed with EdU, and then pulsed with BrdU; sequential pulses of EdU, followed by BrdU, are done without removing or washing out EdU. Curr. Protoc. Cytom. 55:7.38.1‐7.38.15. © 2011 by John Wiley & Sons, Inc.