Single-stranded DNA (ssDNA) is an important intermediate in many DNA repair pathways. Here we describe protocols that permit the measurement of ssDNA that has arisen in the yeast genome in vivo, in response to telomere uncapping. Yeast strains defective in DNA damage response (DDR) genes can be used to infer the roles of the corresponding proteins in regulating ssDNA production and in responding to ssDNA. Using column based methods to purify yeast genomic DNA and quantitative amplification of single-stranded DNA (QAOS) it is possible to measure ssDNA at numerous single copy loci in the yeast genome. We describe how to measure ssDNA in synchronous cultures of cdc13-1 mutants, containing a temperature sensitive mutation in an essential telomere capping protein, and in asynchronous cultures of yku70Delta mutants also defective in telomere capping.