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Summary: Biosafety level 3 decontamination precautions motivate measuring microbial colonies using consumable photography instead of expensive automated plate counters or smartphones, and assaying drug treatments–with multiple concentrations per treatment, replicates, and controls–produces hundreds of images. Here, we present a protocol for semi-automated image analysis by hand-tuning three parameters. The parameters control for non-uniform colony growth and artifacts such as lid condensation, reflections, and plating streaks. We describe steps to prepare images, tune parameters, and plot dose–response relationships.For complete details on the use and execution of this protocol, please refer to Larkins-Ford et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.