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One of the most used and versatile methods to study number, dimensions, content and localization of secretory organelles is confocal microscopy analysis. However, considerable heterogeneity exists in the number, size and shape of secretory organelles that can be present in the cell. One thus needs to analyze large numbers of organelles for valid quantification. Properly evaluating these parameters requires an automated, unbiased method to process and quantitatively analyze microscopy data. Here, we describe two pipelines, run by CellProfiler software, called OrganelleProfiler and OrganelleContentProfiler. These pipelines were used on confocal images of endothelial colony forming cells (ECFCs), which contain unique secretory organelles called Weibel-Palade bodies (WPBs), and on early endosomes in ECFCs and human embryonic kidney 293T (HEK293T) cells. Results show that the pipelines can quantify the cell count, size, organelle count, organelle size, shape, relation to cells and nuclei, and distance to these objects in both endothelial and HEK293T cells. Additionally, the pipelines were used to measure the reduction in WPB size after disruption of the Golgi and to quantify the perinuclear clustering of WPBs after triggering of cAMP-mediated signaling pathways in ECFCs. Furthermore, the pipeline is able to quantify secondary signals located in or on the organelle or in the cytoplasm, such as the small WPB GTPase Rab27A. Cell profiler measurements were checked for validity using Fiji. To conclude, these pipelines provide a powerful, high-processing quantitative tool for the characterization of multiple cell and organelle types. These pipelines are freely available and easily editable for use on different cell types or organelles., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Laan et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)