The combination of mass cytometry and immunohistochemistry (IHC) enables new histopathological imaging methods in which dozens of proteins and protein modifications can be visualized simultaneously in a single tissue section. The power of multiplexing combined with spatial information and quantification was recently illustrated on breast cancer tissue and was described as next‐generation IHC. Robust, accurate, and high‐throughput cell segmentation is crucial for the analysis of this new generation of IHC data. To this end, we propose a watershed‐based cell segmentation, which uses a nuclear marker and multiple membrane markers, the latter automatically selected based on their correlation. In comparison with the state‐of‐the‐art segmentation pipelines, which are only using a single marker for object detection, we could show that the use of multiple markers can significantly increase the segmentation power, and thus, multiplexed information should be used and not ignored during the segmentation. Furthermore, we provide a novel, user‐friendly open‐source toolbox for the automatic segmentation of multiplexed histopathological images. © 2015 International Society for Advancement of Cytometry