Telomere replication is a critical process for preserving genome integrity. The telomere replication fork proceeds unidirectionally from the last subtelomeric origin towards the end of the chromosome, replicating the 5′–3′ G-rich strand by lagging mechanisms and the complementary C-rich strand by leading mechanisms. It has been proposed that the G-rich nature of telomeres may favor the formation of secondary structures such as G-quadruplexes during replication and that specific mechanisms must prevent this to allow the fork to progress unimpeded. The potential of G-quadruplex formation by telomeric sequences has been clearly demonstrated in vitro but it is not known whether these structures form in vivo. We tested the effect of a potent and specific G-quadruplex ligand, telomestatin (TMS), on telomere replication using a novel quantitative approach applied to CO-FISH. We show that TMS, although it penetrates and persists within cells, does not affect telomere replication after short or long-term treatments of mouse embryonic fibroblasts. It does however affect the hybridization efficiency of FISH telomeric probes that recognize the G-rich strand. Our work illustrates the use of a novel technique to measure telomere replication efficiency and suggests that G-quadruplex ligands do not affect telomere replication in a non tumoral context.