Antibiotics are required to treat a wide variety of microbial infections. However, with continued use of antibiotics, pathogens have developed antibiotic resistance that has rendered some antibiotics ineffective. Mtb, the microbe that causes tuberculosis, is particularly deadly, killing approximately 1.1 million people per year. Like other bacteria, Mtb has evolved mechanisms to resist the effects antibiotics. Understanding how antibiotic resistance emerges is critical for the development of countermeasures to battle emerging antibiotic resistance. We developed a technique named One-Cell Doubling Evaluation of Living Arrays of Mycobacterium, or ODELAM, that uses a microscope to automatically watch tens of thousands of individual Mtb cells grow over time as they are exposed to antibiotics. Remarkably, although cells are genetically identical, individual cells in a population respond differently to antibiotic drugs. This so-called antibiotic-induced population heterogeneity is key to how organisms like Mtb develop antibiotic resistance. The detailed growth measurements that ODELAM provides offer insight into the mechanisms of how antibiotics kill Mtb and what strategies Mtb develops to become resistant to antibiotics. The sensitivity of ODELAM allows us to detect different resistance states in individual clinical isolates. This is critical for detecting very small populations of resistance that can persist despite drug treatment. We anticipate these investigations will provide additional information for the design of new antibiotics and inform more effective application of antibiotics to patients.