A novel monochrome multiplex quantitative PCR (MMQPCR) method for measuring telomere length is described. This method allows simultaneous measurement of telomere (T) and single copy gene (S) signals in the same reaction wells, eliminating the need for separate wells for T and S signals. This approach reduces variability due to DNA pipetting and increases throughput while lowering costs. The method uses a single fluorescent DNA-intercalating dye, as T signals can be collected early in the reaction before S signals rise, and S signals can be collected at a temperature that fully melts the telomere product, sending its signal to baseline. The correlation between T/S ratios and Terminal Restriction Fragment (TRF) lengths measured by Southern blot was stronger with this method (R² = 0.844) compared to the original singleplex method (R² = 0.677). Multiplex T/S results from independent runs were highly reproducible (R² = 0.91). The method involves designing primers that generate fixed-length products for telomere repeats and single copy genes. Telomere primers (telg and telc) generate a short, fixed-length product, while single copy gene primers (albumin and beta-globin) are designed to melt at higher temperatures, allowing separation of signals. The thermal cycling profile includes steps that allow the collection of T and S signals at different temperatures, ensuring accurate quantification. The method was validated using 95 DNA samples, showing strong correlation between T/S ratios and TRF lengths (R² = 0.844). The T/S ratios were highly reproducible across independent runs (R² = 0.91) and were independent of the single copy gene used (R² = 0.934). The MMQPCR method is more accurate and reproducible than the singleplex method and can be adapted for many pairs of DNA templates with different copy numbers. It also avoids the need for expensive multi-color fluorescent probes. The method is expected to be useful for studying telomere length and other DNA templates in epidemiological studies.A novel monochrome multiplex quantitative PCR (MMQPCR) method for measuring telomere length is described. This method allows simultaneous measurement of telomere (T) and single copy gene (S) signals in the same reaction wells, eliminating the need for separate wells for T and S signals. This approach reduces variability due to DNA pipetting and increases throughput while lowering costs. The method uses a single fluorescent DNA-intercalating dye, as T signals can be collected early in the reaction before S signals rise, and S signals can be collected at a temperature that fully melts the telomere product, sending its signal to baseline. The correlation between T/S ratios and Terminal Restriction Fragment (TRF) lengths measured by Southern blot was stronger with this method (R² = 0.844) compared to the original singleplex method (R² = 0.677). Multiplex T/S results from independent runs were highly reproducible (R² = 0.91). The method involves designing primers that generate fixed-length products for telomere repeats and single copy genes. Telomere primers (telg and telc) generate a short, fixed-length product, while single copy gene primers (albumin and beta-globin) are designed to melt at higher temperatures, allowing separation of signals. The thermal cycling profile includes steps that allow the collection of T and S signals at different temperatures, ensuring accurate quantification. The method was validated using 95 DNA samples, showing strong correlation between T/S ratios and TRF lengths (R² = 0.844). The T/S ratios were highly reproducible across independent runs (R² = 0.91) and were independent of the single copy gene used (R² = 0.934). The MMQPCR method is more accurate and reproducible than the singleplex method and can be adapted for many pairs of DNA templates with different copy numbers. It also avoids the need for expensive multi-color fluorescent probes. The method is expected to be useful for studying telomere length and other DNA templates in epidemiological studies.