Ploidy, representing the number of homologous chromosome sets, can be estimated from flow cytometry data acquired on cells stained with a fluorescent DNA dye. This estimation relies on a combination of tools that often require scripting, individual sample curation, and additional analyses. To automate the ploidy estimation for multiple flow cytometry files, we developed MuPET-Flow—a Shiny graphical user interface tool. MuPET-Flow allows users to visualize cell fluorescence histograms, detect the peaks corresponding to the different cell cycle phases, perform a linear regression using standards, make ploidy or genome size predictions, and export results as figures and table files. The tool was benchmarked with known ploidy datasets, yielding consistent ploidy results. MuPET-Flow produces ploidy results comparable to those from other available tools; however, MuPET-Flow stands out for its user-friendly interface, which allows the whole process to be carried out in one place, considerably speeding up analysis, particularly in projects involving large numbers of samples. Availability and implementation: https://github.com/CintiaG/MuPET-Flow .