Accurate assays for counting mammalian cells are important tools when performing experiments in cell biology. In general, commercially available cell-counting assays suffer from low throughput and high costs of consumables. Here, we describe a cost-efficient Hoechst and propidium iodide (PI)-staining protocol that accurately counts the number of viable mammalian cells in 96-well microplates based on image cytometry on a Celígo® Image Cytometer. Hands-on time for one microplate is less than 20 minutes and the entire procedure including incubation takes approximately 1 hour. Since all steps prior to image cytometry analysis involve liquid handling and since the Celígo® Image Cytometer is automation-friendly, this protocol has the potential to be integrated into automated liquid handling workflows.