Flow cytometry is an essential tool to monitor DNA content and determine cell cycle distribution. Its utility reflects the relative ease of sample preparation and the stochiometric nature of the most popular DNA-binding dyes (propidium iodide and Sytox Green). Mammalian precedents using flow cytometry for replication and cell biology studies are attractive examples for S. pombe researchers. However, the study of DNA replication with multicolor analysis has lagged behind that in mammalian cells. We present basic and advanced protocols for analysis of DNA replication in fission yeast by flow cytometry including whole cell, nuclear "ghosts," and two-color imaging with BrdU.