The quantitative determination of telomere length has high practical value for the diagnosis and monitoring of oncological and autoimmune diseases. One of the techniques that can be used to measure telomere length is quantitative fluorescence in situ hybridization (Q-FISH). Q-FISH consists of the in situ hybridization of peptide nucleic acid (PNA) probes with metaphase chromosomes or interphase nuclei, followed by digital recording of the fluorescence signal and its quantification using suitable software. However, the accuracy of telomere length measurements depends on several factors, including the properties of the device used, the exposure time, influences of background autofluorescence, and so on. Here, we present the most important steps of Q-FISH image processing and approaches to minimize the influence of confounding factors.