•The C-Circle Assay (CCA) is a rapid, robust and quantitative measure for ALT activity.•Here we detail the protocols and considerations required to perform the CCA.•Including exonuclease removal of linear telomeric DNA and synthesis of C-Circle, C96.•The Quick C-Circle Preparation (QCP; a one tube preparation of cells for the CCA).•Modulation of ALT activity by γ-irradiation. The C-Circle Assay has satisfied the need for a rapid, robust and quantitative ALT assay that responds quickly to changes in ALT activity. 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.