Telomeres are the complex end structures that confer functional integrity and positional stability to human chromosomes. Telomere research has long been dominated by length measurements and biochemical analyses. Recently, interest has shifted towards the role of their three-dimensional organization and dynamics within the nuclear volume. In the mammalian interphase nucleus, there is increasing evidence for a telomeric configuration that is non-random and is cell cycle and cell type dependent. This has functional implications for genome stability. Objective and reproducible representation of the spatiotemporal organization of telomeres, under different experimental conditions, requires quantification by reliable automated image processing techniques. In this paper, we describe methods for quantitative telomere analysis in cell nuclei of living human cells expressing telomere-binding fusion proteins. We present a toolbox for determining telomere positions within the nucleus with subresolution accuracy and tracking telomeres in 4D controlled light exposure microscopy (CLEM) recordings. The use of CLEM allowed for durable imaging and thereby improved segmentation performance considerably. With minor modifications, the underlying algorithms can be expanded to the analysis of other intranuclear features, such as nuclear bodies or DNA double stranded break foci.