The [[C-Circle]] Assay involves (i) extraction or simple preparation (Quick C-Circle Preparation) of the cell's DNA, which includes C-Circles (ii) amplification of the self-primed C-Circles with a rolling circle amplification reaction and (iii) sequence specific detection of the amplification products by native telomeric DNA dot blot or telomeric qPCR. Here we detail the protocols and considerations required to perform the C-Circle Assay and its controls, which include exonuclease removal of linear telomeric DNA, production of the synthetic C-Circle C96 and modulation of ALT activity by γ-irradiation ((Henson JD, Lau LM, Koch S, Martin La Rotta N, Dagg RA, Reddel RR. The C-Circle Assay for alternative-lengthening-of-telomeres activity. Methods. 2017 Feb 1;114:74-84. doi: 10.1016/j.ymeth.2016.08.016. Epub 2016 Sep 3. PMID: 27595911.)).