Alternative lengthening of telomeres (ALT) is likely to be an important target for anticancer treatment as ∼10% of cancers depend on this telomere maintenance mechanism for continued growth, and inhibition of ALT can cause cellular senescence. However, no ALT inhibitors have been developed for therapeutic use because of the lack of a suitable ALT activity assay and of known ALT-specific target molecules. Here we show that partially single-stranded telomeric (CCCTAA)_n DNA circles (C-circles) are ALT specific. We provide an assay that is rapidly and linearly responsive to ALT activity and that is suitable for screening for ALT inhibitors. We detect C-circles in blood from ALT⁺ osteosarcoma patients, suggesting that the C-circle assay (CC assay) may have clinical utility for diagnosis and management of ALT⁺ tumors.