Here we provide a Fiji (ImageJ) macro and a protocol for the automated high-throughput quantification of image-based Live Dead Assays. The macro is free to use and the protocol contains information about the image requirements, step-by-step instructions, and a validation part so you can better interpret the analysis results. In short, the macro batch-processes multi-channel images with a live and a dead fluorescence staining, calculates the resulting cell viability, and saves all information as a data table as well as providing validation overlays for each analyzed image. With this tool it should be possible to quantify large amounts of image data and get reliable and comparable results in a short period of time. We also provide some example images so that you can test the application and performance of the macro. If the macro is of any use for your research, we would highly appreciate a citation of this tool.