Single-stranded DNA (ssDNA) is a DNA repair, replication, and recombination intermediate and a stimulus for checkpoint kinase-dependent cell cycle arrest. Current assays to detect ssDNA generated in vivo are indirect, laborious, and generally require the use of radioactivity. Here, we describe simple, quantitative approaches to measure ssDNA generated in yeast, at single- and multi-copy chromosomal loci and in highly repetitive telomeric sequences. We describe a fluorescence in-gel assay to measure ssDNA in the telomeric TG repeats of telomere cap-defective budding yeast yku70 ∆ and cdc13-1 mutants. We also describe a rapid method to prepare DNA for Quantitative Amplification of ssDNA, used to measure ssDNA in single-copy and repetitive sub-telomeric loci. These complementary methods are useful to understand the important roles of ssDNA in yeast cells and could be readily extended to other cell types.