Telomeres terminate in 3' single-stranded G-overhangs that function in telomere end protection and telomerase action. An accurate measurement of overhang length is challenging due to the presence of many kilobases of double-stranded telomere DNA. Here, a simple method is described that utilizes duplex-specific nuclease (DSN) to digest all genomic DNA including telomeres, leaving the single-stranded overhangs intact. The telomere single-strand G-rich overhang length can then be determined by Southern blot-based assays.