Understanding telomere biology is of utmost importance for aging and cancer research. An essential tool is the determination of telomere length, which traditionally is done by telomere restriction fragment analysis, a laborious and time consuming method. Therefore, large efforts have been made to establish alternative methods like flow FISH analysis. This method, combining fluorescence in situ hybridization with a telomere specific peptide nucleic acid probe and flow cytometry, measures single cells, is suitable for analysis of non-dividing cells, and can be performed within 24 h. However, when performing flow FISH analysis with normal human kidney epithelial cells, we observed strong variation of autofluorescence at different population doubling levels, especially at replicative senescence, which limits the suitability of this method for the analysis of normal human cells. Since molecules responsible for autofluorescence are predominantly accumulating in the cytoplasm, we decided to isolate the nuclei to perform flow FISH analysis. With this novel nuclear flow FISH (NFF) technique we were able to minimize autofluorescence and its variability, thereby improving the signal-to-noise ratio and consequently, allowing the determination of telomere length during in vitro aging with high accuracy. Moreover, NFF will find broader applications, whenever in situ hybridization signals have to be quantitated.