The sensitive telomeric repeat amplification protocol (TRAP) permits telomerase detection in mammalian cell and tissue extracts with very low telomerase activity levels. Unfortunately, conventional TRAP assays require complex post‐amplification procedures, such as polyacrylamide gel electrophoresis and densitometry, to measure telomerase products. Therefore, a real‐time quantitative TRAP assay (RQ‐TRAP) was optimized in the present study and evaluated in comparison with a commercially available quantitative TRAP kit and by monitoring telomerase activity in human hepatocyte cultures, human hepatoma cell lines and telomerase reconstitution experiments. The novel real‐time telomerase detection method has many advantages. Other than sample extraction and real‐time cycling, no additional time‐consuming steps have to be performed for telomerase quantification; reliable and linear telomerase quantification is possible down to single‐cell dilutions without the interference of primer‐dimer artifacts, and the costs are less. Moreover, the precision is similar to other amplification‐based telomerase quantification assays and the results are comparable to data obtained with two commercially available assays. The closed‐tube system reduces the risk of carryover contamination and supports high throughput. In conclusion, RQ‐TRAP provides a new tool for the rapid and reliable quantification of telomerase activity.