Ca2+ microdomains play a key role in intracellular signaling processes. For instance, they mediate the activation of T cells and, thus, the initial adaptive immune system. They are, however, also of utmost importance for activation of other cells, and a detailed understanding of the dynamics of these spatially localized Ca2+ signals is crucial for a better understanding of the underlying signaling processes. A typical approach to analyze Ca2+ microdomain dynamics is live cell fluorescence microscopy imaging. Experiments usually involve imaging a larger number of cells of different groups (for instance, wild type and knockout cells), followed by a time consuming image and data analysis. With DARTS, we present a modular Python pipeline for efficient Ca2+ microdomain analysis in live cell imaging data. DARTS (Deconvolution, Analysis, Registration, Tracking, and Shape normalization) provides state-of-the-art image postprocessing options like deep learning-based cell detection and tracking, spatio-temporal image deconvolution, and bleaching correction. An integrated automated Ca2+ microdomain detection offers direct access to global statistics like the number of microdomains for cell groups, corresponding signal intensity levels, and the temporal evolution of the measures. With a focus on bead stimulation experiments, DARTS provides a so-called dartboard projection analysis and visualization approach. A dartboard projection covers spatio-temporal normalization of the bead contact areas and cell shape normalization onto a circular template that enables aggregation of the spatiotemporal information of the microdomain detection results for the individual cells of the cell groups of interest. The dartboard visualization allows intuitive interpretation of the spatio-temporal microdomain dynamics at the group level. The application of DARTS is illustrated by three use cases in the context of the formation of initial Ca2+ microdomains after cell stimulation. DARTS is provided as an open-source solution and will be continuously extended upon the feedback of the community.