Germination is an important developmental process that supports resumption of growth in dormant spores. The study of the mechanisms underlying germination requires a pure spore population devoid of other cell types. This article describes the sporulation of wild Saccharomyces cerevisiae and Saccharomyces paradoxus strains, and the isolation and purification of ascospores. We also describe a method to synchronously induce germination in a spore population as well as to measure spore activation. This procedure can be applied, for example, to the study of environmental conditions that trigger germination. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Sporulation Basic Protocol 2: Spore purification Basic Protocol 3: Germination induction Support Protocol 1: Flow cytometry analysis Support Protocol 2: Heat-shock resistance measurement.