Yeast chronological life span (CLS) is defined as the capacity of stationary cultures to maintain viability over time, thus mimicking the situation of post-mitotic cells in multicellular organisms. Cellular viability is typically determined by using the colony formation unit assay (CFU). CFU counting is simple but laborious and does not accommodate large-scale experiments. Importantly, viability is determined on the basis of the cell's ability to divide and form a colony, probably not the ideal parameter when studying post-mitotic cellular ageing. This study describes the optimization and validation of a method based on the flow cytometric monitoring of propidium iodide (PI) uptake for assessing yeast cell death during CLS. The optimized protocol is quick, reliable, reproducible and can accommodate high-throughput studies. The method was validated by determining CLS of several strains used in yeast ageing research and by evaluating the effect of genetic disturbances known to extend or reduce yeast chronological life span.